Influenza virus vaccines and uses thereof

ABSTRACT

Provided herein are isolated mutant influenza hemagglutinin polypeptides, methods for providing isolated mutant hemagglutinin polypeptides, compositions comprising the same, vaccines comprising the same and methods of their use, in particular in the detection, prevention and/or treatment of influenza.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made, at least in part, with Government support underAgreement HHSO10020170018C, awarded by HHS. The Government has certainrights in the invention.

INTRODUCTION

The invention relates to the field of medicine. Provided herein areisolated influenza hemagglutinin polypeptides, methods for providinghemagglutinin polypeptides, compositions comprising the same, vaccinescomprising the same and methods of their use, in particular in thedetection, prevention and/or treatment of influenza.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “688097.562US Sequence Listing” and a creation date of Sep.16, 2019 and having a size of 468 kb. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Influenza A and B viruses are major human pathogens, causing arespiratory disease (commonly referred to as “influenza” or “the flu”)that ranges in severity from sub-clinical infection to primary viralpneumonia which can result in death. The WHO estimates that annualepidemics of influenza result in ˜1 billion infections, 3-5 millioncases of severe illness and 300,000-500,000 deaths. The severity ofpandemic influenza depends on multiple factors, including the virulenceof the pandemic virus strain and the level of pre-existing immunity. Themost severe influenza pandemic, in 1918, resulted in >40 million deathsworldwide. Influenza vaccines are formulated every year to match thecirculating strains, as they evolve antigenically owing to antigenicdrift. Nevertheless, vaccine efficacy is not optimal and is dramaticallylow in the case of an antigenic mismatch between the vaccine and thecirculating virus strain. Antiviral agents that target the influenzavirus enzyme neuraminidase have been developed for prophylaxis andtherapy. However, the use of these antivirals is still limited. Emergingapproaches to combat influenza include the development of universalinfluenza virus vaccines that provide protection against antigenicallydistant influenza viruses (Krammer et al., Nat. Rev. Disease Primers 4:3(2018)).

During the last three decades two distinct influenza B lineages haveco-circulated in the population to a varying extent each season, and thedominant B lineage in a specific season has proved hard to predict,complicating the decision of which lineage to include in the trivalentvaccine (TIV) (Ambrose et al., Hum. Vaccin. Immunother. 8:81-8 (2012);US Centers for Disease Control and Prevention, “Seasonal influenzaactivity surveillance reports 2001-2018”www.cdc.gov/flu/weekly/pastreports.htm (accessed on Jul. 2, 2018);European Centre for Disease Prevention and Control/WHO Regional Officefor Europe, “Annual epidemiological reports on seasonal influenza2001-2018,”ecdc.europa.eu/en/seasonal-influenza/surveillance-and-disease-data/aer(accessed on Jul. 2, 2018)). The importance of an effective coverage ofinfluenza B by vaccination is demonstrated by its contribution to theoverall burden of seasonal influenza. According to data from the USCenters for Disease Control, and reports from several Europeancountries, influenza B was responsible for 0.8-82% of the totallaboratory confirmed influenza cases between 2001 and 2018 with aseasonal average of 25% (Ambrose et al., Hum. Vaccin. Immunother. 8:81-8(2012); US Centers for Disease Control and Prevention, “Seasonalinfluenza activity surveillance reports 2001-2018”www.cdc.gov/flu/weekly/pastreports.htm (accessed on Jul. 2, 2018);European Centre for Disease Prevention and Control/WHO Regional Officefor Europe, “Annual epidemiological reports on seasonal influenza2001-2018,”ecdc.europa.eu/en/seasonal-influenza/surveillance-and-disease-data/aer(accessed on Jul. 2, 2018); Dijkstra et al., Epidemiol. Infect.137:473-9 (2009); Peltola et al., Clin. Infect. Dis. 36:299-305 (2003)).Moreover, influenza B is a major contributor to the total morbidity andmortality from influenza, with attributable hospitalization rate similarto influenza A/H3N2 and greater than A/H1N1 (Thompson et al., JAMA292:1333-40 (2004)), accounting for 15% of all influenza attributablerespiratory and circulatory-related death in the United States and 34%among paediatric patients (Ambrose et al., Hum. Vaccin. Immunother.8:81-8 (2012); Thompson et al., JAMA 289:179-86 (2003)). Theseprinciples prompted several health authorities, including the WorldHealth Organization and the US Advisory Committee on ImmunizationPractices, to recommend quadrivalent influenza vaccine (QIV) containingtwo influenza B antigens (one of each B lineage) as one of the optionsfor seasonal vaccination (Grohskopf et al., MMWR Recomm. Rep. 66:1-20(2017); Grohskopf et al., MMWR Recomm. Rep. 67:643-5 (2018); WorldHealth Organization, “Recommended composition of influenza virusvaccines for use in the 2017-2018 northern hemisphere influenza season,”www.whaint/influenza/vaccines/virus/recommendations/2018_19_north/en(accessed on Jul. 2, 2018)).

The current immunization practice relies on early identification ofcirculating influenza viruses to allow for timely production of aneffective seasonal influenza vaccine. Apart from the inherentdifficulties in predicting the strains that will be dominant during thenext season, antiviral resistance and immune escape also play a role infailure of current vaccines to prevent morbidity and mortality. Inaddition to this the possibility of a pandemic caused by a highlyvirulent viral strain originating from animal reservoirs and reassortedto increase human to human spread, poses a significant and realisticthreat to global health.

Influenza type B virus strains are almost exclusively found in humans.The antigenic variation in HA within the influenza type B virus strainsis smaller than those observed within the type A strains. Twogenetically and antigenically distinct lineages of influenza B virus arecirculating in humans, as represented by the B/Yamagata/16/88 (alsoreferred to as B/Yamagata) and B/Victoria/2/87 (B/Victoria) lineages(Ferguson et al., 2003). Although the spectrum of disease caused byinfluenza B viruses is generally milder than that caused by influenza Aviruses, severe illness requiring hospitalization is still frequentlyobserved with influenza B infection.

It is known that antibodies that neutralize the influenza virus areprimarily directed against hemagglutinin (HA). Hemagglutinin or HA is atrimeric glycoprotein that is anchored to the viral coat and has a dualfunction: it is responsible for binding to the cell surface receptorsialic acid and, after uptake, it mediates the fusion of the viral andendosomal membrane leading to release of the viral RNA in the cytosol ofthe cell. HA comprises a large head domain and a smaller stem domain.Attachment to the viral membrane is mediated by a C-terminal anchoringsequence connected to the stem domain. The protein ispost-translationally cleaved in a designated loop to yield twopolypeptides, HA1 and HA2 (the full sequence is referred to as HA0). Themembrane distal head region is mainly derived from HA1 and the membraneproximal stem region primarily from HA2.

The reason that the seasonal influenza vaccine must be updated everyyear is the large variability of the virus. In the hemagglutininmolecule this variation is particularly manifested in the head domainwhere antigenic drift and shift have resulted in a large number ofdifferent variants. Since this is also the area that is immunodominant,most neutralizing antibodies are directed against this domain and act byinterfering with receptor binding. The combination of immunodominanceand large variation of the head domain also explains why infection witha particular strain does not lead to immunity to other strains: theantibodies elicited by the first infection only recognize a limitednumber of strains closely related to the virus of the primary infection.

Thus, there is a need for developing a universal influenza virus vaccinethat stimulates the production of a robust, broadly protective responseagainst current and future influenza virus strains (both seasonal andpandemic), in particular, providing protection against the influenza Bvirus for effective prevention and therapy of influenza.

SUMMARY

Provided herein are isolated mutant influenza hemagglutininpolypeptides, methods for providing the isolated hemagglutininpolypeptides, compositions comprising the same, vaccines comprising thesame, and methods of using the compositions and vaccines.

Provided herein are isolated mutant influenza hemagglutininpolypeptides. The isolated mutant influenza hemagglutinin polypeptidescomprise at least two stabilizing mutations in the polypeptide, whereinthe stabilizing mutations comprise substitution mutations at (a) aminoacid positions 227 and/or 238; and/or (b) amino acid positions 384and/or 476, wherein the amino acid position corresponds to the aminoacid position of SEQ ID NO:1. In certain embodiments, (a) amino acidposition 227 is substituted with an amino acid selected from the groupconsisting of Q, N, F, I, and Y, and/or amino acid position 238 issubstituted with an amino acid selected from the group consisting of N,Q, I, and F; and/or (b) amino acid position 384 is substituted with anamino acid selected from the group consisting of W, F, N, Q, and I,and/or amino acid position 476 is substituted with an amino acidselected from the group consisting of W, F, Y, I, N, and Q. In certainembodiments, (a) amino acid position 227 is substituted with a Q andamino acid position 238 is substituted with an I; and/or (b) amino acidposition 384 is substituted with an I and amino acid position 476 issubstituted with an I. In certain embodiments, the isolated mutantinfluenza hemagglutinin polypeptide further comprises one additionalstabilizing mutation in the polypeptide. The additional stabilizingmutation is a substitution at amino acid position 461, wherein the aminoacid position corresponds to the amino acid position in SEQ ID NO:1. Incertain embodiments, amino acid position 461 is substituted with anamino acid selected from the group consisting of M, L, W, Y, and R. Incertain embodiments, amino acid position 461 is substituted with an R.The isolated mutant influenza hemagglutinin polypeptide can, forexample, comprise an amino acid sequence selected from SEQ ID NO:19, SEQID NO:35, SEQ ID NO:39 or SEQ ID NO:40. The isolated mutant influenzahemagglutinin polypeptide can, for example, comprise an amino acidsequence of SEQ ID NO:8.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide further comprises at least one additional glycan motif in ahead domain of the polypeptide. The glycan motif can, for example,comprise a substitution of an amino (N)-linked glycosylation motif in atleast one amino acid position selected from the group consisting of (a)136 or 137, (b) 141, and (c) 151, wherein the amino acid positioncorresponds to the amino acid position of SEQ ID NO:1. The glycan motifcan, for example, comprise a substitution of the N-linked glycosylationmotif at amino acid positions 136 and 141, 136 and 151, 137 and 141, 137and 151, or 141 and 151. In certain embodiments, the glycan motifcomprises the substitution of the N-linked glycosylation motif at aminoacid positions 141 and 151. In certain embodiments, the mutant influenzahemagglutinin polypeptide comprises an amino acid sequence selected fromSEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide further comprises or solely comprises a receptor bindingsite mutation in the polypeptide. The receptor binding site mutationcan, for example, comprise a substitution at an amino acid positionselected from the group consisting of (a) 175, (b) 219, (c) 257, and (d)258, wherein the amino acid position corresponds to the amino acidposition of SEQ ID NO:1. In certain embodiments, (a) 175 is substitutedwith an amino acid selected from the group consisting of F, W, and Y;(b) 219 is substituted with an amino acid selected from the groupconsisting of F, W, Y, R, and E; (c) 257 is substituted with an aminoacid selected from the group consisting of E, D, V, F; or (d) 258 issubstituted with an amino acid selected from the group consisting of E,D, V, and F. In certain embodiments, (a) 175 is substituted with a W,(b) 219 is substituted with an E, (c) 257 is substituted with an E, or(d) 258 is substituted with an E. In certain embodiments, the mutantinfluenza hemagglutinin polypeptide comprises an amino acid sequenceselected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, or SEQ ID NO:61.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide further comprises an amino acid substitution at position136, wherein the amino acid position corresponds to the amino acidposition of SEQ ID NO:1.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide, further comprises or solely comprises a fusion peptideproximal region (FPPR) deletion mutation. The FPPR deletion mutationcan, for example, comprise a deletion of at least three to seven aminoacid residues between amino acid position 369 and 382, wherein the aminoacid position corresponds to the amino acid position of SEQ ID NO:1. TheFPPR deletion mutation can, for example, comprise a deletion selectedfrom the group consisting of Δ372-376, Δ372-378, Δ373-377, Δ373-376,Δ374-379, Δ374-376, Δ376-380, and Δ377-381. In certain embodiments, theFPPR deletion mutation is a deletion selected from Δ372-376 or Δ376-380.In certain embodiments, the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:62 or SEQ IDNO:68.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide comprises an amino acid sequence selected from SEQ ID NO:70,SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide can comprise a foldon domain. In certain embodiments, theisolated mutant influenza hemagglutinin polypeptide further comprises acarboxy (C)-terminal truncation starting at an amino acid position fromamino acid 532 to amino acid position 549, wherein the amino acidpositon corresponds to the amino acid position of SEQ ID NO:1. Incertain embodiments, the C-terminal truncation starts at amino acidposition 532, 534, 536, 539, 541, 543, 545, 547, or 549, wherein theamino acid position corresponds to the amino acid position of SEQ IDNO:1.

In certain embodiments, the mutant influenza hemagglutinin polypeptidefurther comprises an amino acid substitution at a cleavage site at aminoacid position 362, wherein wherein the amino acid position correspondsto the amino acid position of SEQ ID NO:1. The cleavage sitesubstitution at amino acid position 362 can, for example, be a Q.

Also provided is an isolated nucleic acid encoding an isolated mutantinfluenza hemagglutinin polypeptide described herein.

Also provided is a vector comprising an isolated nucleic acid describedherein.

Also provided is a host cell comprising a vector described herein.

Also provided is a pharmaceutical composition comprising an isolatedmutant influenza hemagglutinin polypeptide, an isolated mutant influenzahemagglutinin nucleic acid, and/or a vector described herein and apharmaceutically acceptable carrier.

Also provided are methods of inducing an immune response against aninfluenza virus in a subject in need thereof. The methods compriseadministering to the subject in need thereof a pharmaceuticalcomposition described herein.

Also provided are methods of producing an isolated mutant influenzahemagglutinin polypeptide. The methods comprise culturing a host celldescribed herein under conditions capable of producing the mutantinfluenza hemagglutinin polypeptide and recovering the mutant influenzahemagglutinin polypeptide from the cell or culture.

Also provided are methods of producing a pharmaceutical compositiondescribed herein. The methods comprise combining the isolated mutantinfluenza polypeptide with a pharmaceutically acceptable carrier.

The various embodiments and uses of the polypeptides according to theinvention will become clear from the following detailed description ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the structure and design elements of the polypeptidesof the invention. FIG. 1A shows the three-dimensional representation ofthe polypeptides of the invention (representing the ectodomain ofinfluenza B HA; pdb ID 4NRJ, Ni et al., Virology 450-451:71-83 (2014)).FIG. 1B shows a schematic drawing of a certain polypeptide of theinvention UFV180846 (SEQ ID NO:2) with the positions of thesubstitutions indicated; * introduction of N-linked glycosylationmotifs, ¥ fusion peptide proximal region (FPPR) deletion: residues372-376 are omitted, ϕ C-terminus truncated in this example afterresidue 536 (numbering refers to WT HA; SEQ ID NO:1).

FIGS. 2A-2F show the analysis of EXPI-293 expressed polypeptides withstabilizing mutations, normalized to reference wild type FL HAB/Brisbane/60/08 containing a Foldon trimerization domain (UFV170090)(SEQ ID NO:3). FIG. 2A shows a schematic representation of the monomericHA ectodomain with the positions of the amino acid substitutionsindicated in spheres. Specified are the residues as present in wild type(WT) HA. FIG. 2B shows AlphaLISA binding of monoclonal antibody CR9114to polypeptides of the invention carrying various amino acidsubstitutions at position 461. Binding is shown as a relative % ofrespective reference HA sequence. FIG. 2C shows AlphaLISA binding ofmonoclonal antibody CR9114 to polypeptides of the invention carryingvarious amino acid substitutions at positions 227 and 236. Binding isshown as a relative % of respective reference HA sequence. FIG. 2D showsAlphaLISA binding of monoclonal antibody CR9114 to polypeptides of theinvention carrying various amino acid substitutions at positions 384 and476. Binding is shown as a relative % of respective reference HAsequence. FIG. 2E shows the expression level and CR9114 binding asdetermined by AlphaLISA and temperature stability as determined by DSFof polypeptides with combinations of stabilizing substitutions. Bindingis shown as a relative % of respective reference HA sequence. FIG. 2Fshows SEC profiles of polypeptides; dotted line representing the WT HA(UFV170090) including Foldon trimerization domain. The black linesrepresenting stabilized polypeptides with (UFV170525 (SEQ ID NO:19), andUFV170556 (SEQ ID NO:35)) and without (UFV171348 (SEQ ID NO:39) andUFV171387 (SEQ ID NO:40)) a foldon trimerization domain. The ‘−’ symbolas present in FIGS. 2B, 2C, 2D, and 2E indicates WT residues are presentat position indicated in the column header. In FIG. 2E, the ‘+’ and ‘−’symbol in the Foldon column indicate the presence or absence of theC-terminal foldon trimerization domain, respectively.

FIGS. 3A-3B show the analysis of EXPI-293 expressed polypeptides withintroduced amino (N)-linked glycosylation motifs in the head domain.FIG. 3A shows a schematic representation of the wild type monomeric HAwith the positions of the point substitutions indicated in spheres. FIG.3B shows the protein expression levels, trimer content, and antibodybinding as determined by AlphaLISA. Values are normalized to referencepolypeptide UFV171990 (SEQ ID NO:41) for UFV171991 (SEQ ID NO:45),UFV171992 (SEQ ID NO:44), and UFV171993 (SEQ ID NO:42); and referencepolypeptide UFV170090 (SEQ ID NO:3) for UFV171472 (SEQ ID NO:42). The‘+’ symbol indicates the presence of an N-linked glycosylation motif atthe particular position. Symbol F indicates the presence of Foldontrimerization domain.

FIGS. 4A-4B show the analysis of EXPI-293 expressed polypeptides withintroduced mutations near the receptor binding site. FIG. 4A shows aschematic representation of monomeric HA with positions of the pointsubstitutions indicated in spheres. FIG. 4B shows the protein expressionlevels, trimer content, and antibody binding as determined by AlphaLISA.Values are normalized to reference polypeptide UFV171990 (SEQ ID NO:41).The ‘−’ symbol indicates the particular position is not mutated and theWT residue is present.

FIGS. 5A-5B show the analysis of EXPI-293 expressed polypeptides withdeletions in the Fusion Peptide Proximal Region (FPPR). FIG. 5A shows aschematic representation of the monomeric ectodomain of HA with the areaof the deleted position in the FPPR indicated in black spheres. FIG. 5Bshows the protein expression levels, trimer content, and antibodybinding as determined by AlphaLISA. Values are normalized to referencepolypeptide UFV171990 (SEQ ID NO:41).

FIG. 6 shows SEC profiles of EXPI-293 culture supernatants expressingsoluble trimeric polypeptide variants with alternative C-terminaltruncations (in UFV180454 (SEQ ID NO:71) at position 549 stepwise downto position 532 in UFV180462 (SEQ ID NO:79)); polypeptide (black line)and full-length reference UFV180284 (SEQ ID NO:70) (dotted line) thatincludes a C-tag.

FIGS. 7A-7E show the analysis of EXPI-CHO culture supernatant expressingsoluble polypeptides and in vitro characterization of purifiedpolypeptides. Various combinations of substitutions were evaluated;amino (N)-linked glycan motifs in the head domain, stabilizingsubstitutions, receptor binding site substitution 257E and FPPRdeletions. FIG. 7A shows SEC profiles of supernatant of cells expressingUFV180131 (SEQ ID NO:81) (left panel) and of purified UFV180131 (SEQ IDNO:81) (right panel). FIG. 7B shows the expression level of polypeptidesas determine by OCTET. EC₅₀ values of stem (CR9114), neck (CR8071), andhead domain (SD84) specific antibodies to purified HA as determined byELISA. Temperature stability of purified polypeptides by DifferentialScanning Fluorimetry. The ‘−’ symbol indicates that a particularposition is not mutated and the WT residue is present. FIG. 7C shows theprotein expression levels of construct UFV180846 (SEQ ID NO:84)expressed in EXPI-CHO culture supernatants as determined by OCTET, EC₅₀values of stem (CR9114), neck (CR8071), and head domain (34B5(WO2015/148806)) specific antibodies to purified HA as determined byELISA, and temperature stability of the purified polypeptide byDifferential Scanning Fluorimetry. FIG. 7D shows an alignment of theVictoria lineage (SEQ ID NO:1), the Yamagata lineage (SEQ ID NO:94), theconsensus sequence (SEQ ID NO:95), UFV170088 (SEQ ID NO:80), UFV180131(SEQ ID NO:81), UFV180137 (SEQ ID NO:82), UFV180251 (SEQ ID NO:83), andUFV180284 (SEQ ID NO:). FIG. 7E shows an alignment of the Victorialineage (SEQ ID NO:1), the Yamagata lineage (SEQ ID NO:94), theconsensus sequence (SEQ ID NO:95), UFV170088 (SEQ ID NO:80), UFV180846(SEQ ID NO:84), UFV180847 (SEQ ID NO:91); UFV180848 (SEQ ID NO:92), andUFV180849 (SEQ ID NO:93).

DEFINITIONS

Definitions of terms as used in the present invention are given below.

An amino acid according to the invention can be any of the twentynaturally occurring (or ‘standard’ amino acids) or variants thereof,such as e.g. D-proline (the D-enantiomer of proline), or any variantsthat are not naturally found in proteins, such as e.g. norleucine. Thestandard amino acids can be divided into several groups based on theirproperties. Important factors are charge, hydrophilicity orhydrophobicity, size and functional groups. These properties areimportant for protein structure and protein-protein interactions. Someamino acids have special properties such as cysteine, that can formcovalent disulfide bonds (or disulfide bridges) to other cysteineresidues, proline that forms a cycle to the polypeptide backbone, andglycine that is more flexible than other amino acids. Table 1 shows theabbreviations and properties of the standard amino acids.

The term “amino acid sequence identity” refers to the degree of identityor similarity between a pair of aligned amino acid sequences, usuallyexpressed as a percentage. Percent identity is the percentage of aminoacid residues in a candidate sequence that are identical (i.e., theamino acid residues at a given position in the alignment are the sameresidue) or similar (i.e., the amino acid substitution at a givenposition in the alignment is a conservative substitution, as discussedbelow), to the corresponding amino acid residue in the peptide afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence homology. Sequence homology, includingpercentages of sequence identity and similarity, are determined usingsequence alignment techniques well-known in the art, such as by visualinspection and mathematical calculation, or more preferably, thecomparison is done by comparing sequence information using a computerprogram. An exemplary, preferred computer program is the GeneticsComputer Group (GCG; Madison, Wis.) Wisconsin package version 10.0program, ‘GAP’ (Devereux et al. (1984)).

“Conservative substitution” refers to replacement of an amino acid ofone class is with another amino acid of the same class. In particularembodiments, a conservative substitution does not alter the structure orfunction, or both, of a polypeptide. Classes of amino acids for thepurposes of conservative substitution include hydrophobic (e.g. Met,Ala, Val, Leu), neutral hydrophilic (e.g. Cys, Ser, Thr), acidic (e.g.Asp, Glu), basic (e.g. Asn, Gln, His, Lys, Arg), conformation disrupters(e.g. Gly, Pro) and aromatic (e.g. Trp, Tyr, Phe).

As used herein, the terms “disease” and “disorder” are usedinterchangeably to refer to a condition in a subject. In someembodiments, the condition is a viral infection, in particular aninfluenza virus infection. In specific embodiments, a term “disease”refers to the pathological state resulting from the presence of thevirus in a cell or a subject, or by the invasion of a cell or subject bythe virus. In certain embodiments, the condition is a disease in asubject, the severity of which is decreased by inducing an immuneresponse in the subject through the administration of an immunogeniccomposition.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich has a prophylactic and/or therapeutic effect(s). In certainembodiments, an “effective amount” in the context of administration of atherapy to a subject refers to the amount of a therapy which issufficient to achieve a reduction or amelioration of the severity of aninfluenza virus infection, disease or symptom associated therewith, suchas, but not limited to a reduction in the duration of an influenza virusinfection, disease or symptom associated therewith, the prevention ofthe progression of an influenza virus infection, disease or symptomassociated therewith, the prevention of the development or onset orrecurrence of an influenza virus infection, disease or symptomassociated therewith, the prevention or reduction of the spread of aninfluenza virus from one subject to another subject, the reduction ofhospitalization of a subject and/or hospitalization length, an increaseof the survival of a subject with an influenza virus infection ordisease associated therewith, elimination of an influenza virusinfection or disease associated therewith, inhibition or reduction ofinfluenza virus replication, reduction of influenza virus titer; and/orenhancement and/or improvement of the prophylactic or therapeuticeffect(s) of another therapy. In certain embodiments, the effectiveamount does not result in complete protection from an influenza virusdisease but results in a lower titer or reduced number of influenzaviruses compared to an untreated subject. Benefits of a reduction in thetiter, number or total burden of influenza virus include, but are notlimited to, less severe symptoms of the infection, fewer symptoms of theinfection and a reduction in the length of the disease associated withthe infection.

The term “host,” as used herein, is intended to refer to an organism ora cell into which a vector such as a cloning vector or an expressionvector has been introduced. The organism or cell can be prokaryotic oreukaryotic. Preferably, the host comprises isolated host cells, e.g.host cells in culture. The term “host cells” merely signifies that thecells are modified for the (over)-expression of the polypeptides of theinvention. It should be understood that the term host is intended torefer not only to the particular subject organism or cell but to theprogeny of such an organism or cell as well. Because certainmodifications can occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent organism or cell, but are still included within the scopeof the term “host” as used herein.

The term “included” or “including” as used herein is deemed to befollowed by the words “without limitation.”

As used herein, the term “infection” means the invasion by,multiplication and/or presence of a virus in a cell or a subject. In oneembodiment, an infection is an “active” infection, i.e., one in whichthe virus is replicating in a cell or a subject. Such an infection ischaracterized by the spread of the virus to other cells, tissues, and/ororgans, from the cells, tissues, and/or organs initially infected by thevirus. An infection can also be a latent infection, i.e., one in whichthe virus is not replicating. In certain embodiments, an infectionrefers to the pathological state resulting from the presence of thevirus in a cell or a subject, or by the invasion of a cell or subject bythe virus.

Influenza viruses are classified into influenza virus types: genus A, Band C. The term “subtype” specifically includes all individual“strains,” within each subtype, which usually result from mutations andshow different pathogenic profiles, including natural isolates as wellas man-made mutants or reassortants and the like. Such strains can alsobe referred to as various “isolates” of a viral subtype. Accordingly, asused herein, the terms “strains” and “isolates” can be usedinterchangeably. The current nomenclature for human influenza virusstrains or isolates includes the type (genus) of virus, i.e. A, B or C,the geographical location of the first isolation, strain number and yearof isolation.

As used herein, the term “influenza virus disease” refers to thepathological state resulting from the presence of an influenza virus,e.g. an influenza A or B virus in a cell or subject or the invasion of acell or subject by an influenza virus. In specific embodiments, the termrefers to a respiratory illness caused by an influenza virus.

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid can be single-stranded or double-stranded. The nucleic acidmolecules can be modified chemically or biochemically or can containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those of skill in the art. Such modifications include,for example, labels, methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). A reference to a nucleic acid sequence encompassesits complement unless otherwise specified. Thus, a reference to anucleic acid molecule having a particular sequence should be understoodto encompass its complementary strand, with its complementary sequence.The complementary strand is also useful, e.g., for anti-sense therapy,hybridization probes and PCR primers.

As used herein, in certain embodiments the numbering of the amino acidsin hemagglutinin is based on the numbering of amino acids inhemagglutinin of a wild type influenza virus, e.g. the numbering of theamino acids of the influenza strain B/Brisbane/60/08 (SEQ ID NO: 1). Asused in the present invention, the wording “amino acid position “x” thusmeans the amino acid corresponding to the amino acid at position x inhemagglutinin of the particular wild type influenza virus, e.g.B/Brisbane/60/08 (SEQ ID NO: 1). It will be understood by the skilledperson that equivalent amino acids in other influenza virus strainsand/or subtypes can be determined by multiple sequence alignment. Notethat, in the numbering system used throughout this application 1 refersto the N-terminal amino acid of an immature hemagglutinin protein (SEQID NO: 1). The mature sequence starts e.g. on position 16 of SEQ IDNO: 1. It will be understood by the skilled person that the leadersequence (or signal sequence) that directs transport of a protein duringproduction (e.g. corresponding to amino acids 1-15 of SEQ ID NO: 1),generally is not present in the final polypeptide, that is e.g. used ina vaccine. In certain embodiments, the polypeptides according to theinvention thus comprise an amino acid sequence without the leadersequence, i.e. the amino acid sequence is based on the amino acidsequence of hemagglutinin without the signal sequence.

“Polypeptide” refers to a polymer of amino acids linked by amide bondsas is known to those of skill in the art. As used herein, the term canrefer to a single polypeptide chain linked by covalent amide bonds. Theterm can also refer to multiple polypeptide chains associated bynon-covalent interactions such as ionic contacts, hydrogen bonds, Vander Waals contacts and hydrophobic contacts. Those of skill in the artwill recognize that the term includes polypeptides that have beenmodified, for example by post-translational processing such as signalpeptide cleavage, disulfide bond formation, glycosylation (e.g.,N-linked and O-linked glycosylation), protease cleavage and lipidmodification (e.g. S-palmitoylation).

The term “vector” denotes a nucleic acid molecule into which a secondnucleic acid molecule can be inserted for introduction into a host whereit will be replicated, and in some cases expressed. In other words, avector is capable of transporting a nucleic acid molecule to which ithas been linked. Cloning as well as expression vectors are contemplatedby the term “vector,” as used herein. Vectors include, but are notlimited to, plasmids, cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. Certain vectors are capable of autonomousreplication in a host into which they are introduced (e.g., vectorshaving a bacterial origin of replication can replicate in bacteria).Other vectors can be integrated into the genome of a host uponintroduction into the host, and thereby are replicated along with thehost genome.

As used herein, the term “wild-type” in the context of a virus refers toinfluenza viruses that are prevalent, circulating naturally andproducing typical outbreaks of disease.

As used herein, the term “glycan motif” or “N-linked glycosylationmotif” refers to a specific amino acid motif of a polypeptide, such thatthe specific amino acid motif can be glycosylated through the additionof a glycan molecule. An N-linked glycosylation motif comprises thespecific amino acid motif of NxT/S (wherein x is not a P). In apolypeptide, wherein an N-linked glycosylation motif or glycan motif issubstituted, the amino acid position listed correlates with theasparagine of the NxT/S amino acid motif. By way of an example, in thepolypeptides described below, for positions 136, 137, and 151 an N and Twere introduced into the polypeptide, with the N being introduced atposition 136, 137, and 151 with a threonine being introduced atpositions 138, 139, and 153, respectively, whereas for position 141, anasparagine (N) was present in the wild type sequence, and the motif wascompleted by introducing a threonine at position 143.

DETAILED DESCRIPTION

Influenza viruses have a significant impact on global public health,causing millions of cases of severe illness each year, thousands ofdeaths, and considerable economic losses. Current trivalent andquadrivalent influenza vaccines elicit a potent neutralizing antibodyresponse to the vaccine strains and closely related isolates, but rarelyextend to more diverged strains within a subtype or to other subtypes.In addition, selection of the appropriate vaccine strains presents manychallenges and frequently results in sub-optimal protection.Furthermore, predicting the subtype of the next pandemic virus,including when and where it will arise, is currently impossible.

Hemagglutinin (HA) is the major envelope glycoprotein from influenzaviruses which is the major target of neutralizing antibodies.Hemagglutinin has two main functions during the entry process. First,hemagglutinin mediates attachment of the virus to the surface of targetcells through interactions with sialic acid receptors. Second, afterendocytosis of the virus, hemagglutinin subsequently triggers the fusionof the viral and endosomal membranes to release its genome into thecytoplasm of the target cell. HA comprises a large ectodomain of ˜500amino acids that is cleaved by host-derived enzymes to generate 2polypeptides that remain linked by a disulfide bond. The majority of theN-terminal fragment (HAL 320-330 amino acids) forms a membrane-distalglobular domain that contains the receptor-binding site and mostdeterminants recognized by virus-neutralizing antibodies. The smallerC-terminal portion (HA2, ˜180 amino acids) forms a stem-like structurethat anchors the globular domain to the cellular or viral membrane. Thedegree of sequence homology between HA1 polypeptides is less than thedegress of sequence homology between HA2 polypeptides. The mostconserved region is the sequence around the cleavage site, particularlythe HA2 N-terminal amino acids, which is conserved among all influenza Aand B virus subtypes. Part of this region is exposed as a surface loopin the HA precursor molecule (HA0) but becomes inaccessible when HA0 iscleaved into HA1 and HA2 (Lorieau et al., Proc. Natl. Acad. Aci. USA107:11341 (2010)).

Most neutralizing antibodies bind to the loops that surround thereceptor binding site and interfere with receptor binding andattachment. Since these loops are highly variable, most antibodiestargeting these regions are strain-specific, explaining why currentvaccines elicit such limited, strain-specific immunity. Recently,however, fully human monoclonal antibodies against influenza virushemagglutinin with broad cross-neutralizing potency were generated.Functional and structural analysis have revealed that these antibodiesinterfere with the membrane fusion process and are directed againsthighly conserved epitopes in the stem domain of the influenza HA protein(Throsby et al., 2008; Ekiert et al. 2009, WO 2008/028946,WO2010/130636, WO 2013/007770).

Isolated Mutant Hemagglutinin Polypeptides

According to the present invention new isolated mutant hemagluttininpolypeptides have been designed presenting epitopes for recognition bybroadly protecting antibodies. These polypeptides can be used to createa universal epitope-based vaccine inducing protection against a broadrange of influenza strains. The polypeptides are stabilized and then thehighly variable and immunodominant part, i.e. the head domain, isshielded, immunodampened, through the introduction of glycan molecules.The head can have multiple glycans to shield the epitopes from beingrecognized by the immune system, thus redirecting the immune responsetowards the more conserved neck and stem domain to produce broadlyprotective antibodies.

The isolated mutant hemagluttinin polypeptides of this invention arecapable of presenting the conserved epitopes to the immune system in theabsence of dominant epitopes that are present in the membrane distalhead domain. To this end, part of the primary sequence of thehemagluttinin polypeptide making up the head domain is shielded withglycan molecules. The resulting polypeptide sequence is further modifiedby introducing specific amino acid substitutions that stabilize thenative 3-dimensional structure of the remaining part of thehemagglutinin polypeptide.

According to the invention, the isolated mutant hemagglutininpolypeptides comprise one or more additional mutations, i.e. amino acidsubstitutions and/or glycan motif substitutions, in the head domain, thestem domain, and/or the receptor binding site substitution, as comparedto the amino acid sequence of corresponding wild-type influenza virushemagglutinin polypeptide, i.e. the influenza virus on which the mutanthemagglutinin polypeptides are based.

According to embodiments of the invention, the isolated mutanthemagglutinin polypeptides comprise amino acid substitutions, glycanmotif substitutions, receptor binding site substitutions, and/ordeletion mutations. When referencing the substitutions and deletionmutations, an amino acid position(s) for the substitution(s) and/ordeletion(s) is provided. The amino acid position corresponds to theamino acid sequence of SEQ ID NO:1, as provided herein. By way of anexample, an amino acid substitution at amino acid position 227 wouldcorrespond to an amino acid substitution of the lysine (K) at position227 of SEQ ID NO:1. By way of another example, an amino acidsubstitution at amino acid position 238 would correspond to an aminoacid substitution of the histidine at position 238 of SEQ ID NO:1. Thespecific amino acid position and residue can vary based on the startinghemagglutinin polypeptide sequence of a specific influenza strain;however, one skilled in the art would be capable of performing asequence alignment to identify the corresponding amino acid position andresidue that corresponds to the position on SEQ ID NO:1.

In embodiments of the invention, amino acid substitutions at thespecific amino acid positions will be chosen based on factors whichinclude, but are not limited to, potential for steric hindrance, chargeattraction, charge repulsion, common properties of the amino acid sidechain, secondary and/or tertiary structure considerations, and/orfrequency of use in respective host cells. A person skilled in the artwould understand which factors to consider when designing amino acidsubstitutions for the isolated mutant influenza hemagglutininpolypeptides of the invention.

In certain aspects of the invention, provided herein are isolated mutantinfluenza hemagglutinin polypeptides comprising at least two stabilizingmutations in the polypeptide, wherein the stabilizing mutations comprisesubstitution mutations at (a) amino acid positions 227 and/or 238;and/or (b) amino acid positions 384 and/or 476, wherein the amino acidposition corresponds to the amino acid position of SEQ ID NO:1. Incertain embodiments, (a) amino acid position 227 is substituted with anamino acid selected from the group consisting of Q, N, F, I, and Y,and/or amino acid position 238 is substituted with an amino acidselected from the group consisting of N, Q, I, and F; and/or (b) aminoacid position 384 is substituted with an amino acid selected from thegroup consisting of W, F, N, Q, and I, and/or amino acid position 476 issubstituted with an amino acid selected from the group consisting of W,F, Y, I, N, and Q. In certain embodiments, (a) amino acid position 227is substituted with a Q and amino acid position 238 is substituted withan I; and/or (b) amino acid position 384 is substituted with an I andamino acid position 476 is substituted with an I.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide further comprises one additional stabilizing mutation in thepolypeptide. The additional stabilizing mutation is a substitution atamino acid position 461, wherein the amino acid position corresponds tothe amino acid position in SEQ ID NO:1. In certain embodiments, aminoacid position 461 is substituted with an amino acid selected from thegroup consisting of M, L, W, Y, and R. In certain embodiments, aminoacid position 461 is substituted with an R.

The isolated mutant influenza hemagglutinin polypeptide can, forexample, comprise an amino acid sequence selected from SEQ ID NO:19, SEQID NO:35, SEQ ID NO:39 or SEQ ID NO:40. The isolated mutant influenzahemagglutinin polypeptide can, for example, comprise the amino acidsequence of SEQ ID NO:8 or SEQ ID No: 108.

In certain aspects of the invention, the isolated mutant influenzahemagglutinin polypeptide further comprises at least one additionalglycan motif in a head domain of the polypeptide. The glycan motif can,for example, comprise a substitution of an amino (N)-linkedglycosylation motif in at least one amino acid position selected fromthe group consisting of (a) 136 or 137, (b) 141, and (c) 151, whereinthe amino acid position corresponds to the amino acid position of SEQ IDNO:1. The glycan motif can, for example, comprise a substitution of theN-linked glycosylation motif at amino acid positions 136 and 141, 136and 151, 137 and 141, 137 and 151, or 141 and 151. In certainembodiments, the glycan motif comprises the substitution of the N-linkedglycosylation motif at amino acid positions 141 and 151. In certainembodiments, the mutant influenza hemagglutinin polypeptide comprises anamino acid sequence selected from SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, or SEQ ID NO:45.

In certain aspects of the invention, the isolated mutant influenzahemagglutinin polypeptide further comprises or solely comprises areceptor binding site mutation in the polypeptide. The receptor bindingsite mutation can, for example, comprise a substitution at an amino acidposition selected from the group consisting of (a) 175, (b) 219, (c)257, and (d) 258, wherein the amino acid position corresponds to theamino acid position of SEQ ID NO:1. In certain embodiments, (a) 175 issubstituted with an amino acid selected from the group consisting of F,W, and Y; (b) 219 is substituted with an amino acid selected from thegroup consisting of F, W, Y, R, and E; (c) 257 is substituted with anamino acid selected from the group consisting of E, D, V, F; or (d) 258is substituted with an amino acid selected from the group consisting ofE, D, V, and F. In certain embodiments, (a) 175 is substituted with a W,(b) 219 is substituted with an E, (c) 257 is substituted with an E, or(d) 258 is substituted with an E. In certain embodiments, the mutantinfluenza hemagglutinin polypeptide comprises an amino acid sequenceselected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:55, or SEQ ID NO:61.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide further comprises an amino acid substitution at position136, wherein the amino acid position corresponds to the amino acidposition of SEQ ID NO:1.

In certain aspects of the invention, the isolated mutant influenzahemagglutinin polypeptide, further comprises or solely comprises afusion peptide proximal region (FPPR) deletion mutation. The FPPRdeletion mutation can, for example, comprise a deletion of at leastthree to seven amino acid residues between amino acid position 369 and382, wherein the amino acid position corresponds to the amino acidposition of SEQ ID NO:1. The FPPR deletion mutation can, for example,comprise a deletion selected from the group consisting of Δ372-376,Δ372-378, Δ373-377, Δ373-376, Δ374-379, Δ374-376, Δ376-380, andΔ377-381. In certain embodiments, the FPPR deletion mutation is adeletion selected from Δ372-376 or Δ376-380. In certain embodiments, themutant influenza hemagglutinin polypeptide comprises an amino acidsequence selected from SEQ ID NO:62 or SEQ ID NO:68.

In certain embodiments, the isolated mutant hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:70, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84.

In certain embodiments, the mutant influenza hemagglutinin polypeptidefurther comprises an amino acid substitution at a cleavage site at aminoacid position 362, wherein wherein the amino acid position correspondsto the amino acid position of SEQ ID NO:1. The cleavage sitesubstitution at amino acid position 362 can, for example, be a Q.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide is derived from a hemagglutinin of an influenza B virus. Inparticular, the isolated mutant influenza hemagglutinin polypeptide canbe derived from hemagglutinin of an influenza B virus from theB/Yamagata lineage (as represented by B/Yamagata/16/88) or from theB/Victoria lineage (as represented by B/Victoria/2/87). In certainembodiments, the polypeptide is derived from B/Brisbane/60/08,B/Iowa/06/2017, or B/Lee/40.

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide can comprise a heterologous trimerization domain (e.g., afoldon).

In certain embodiments, the isolated mutant influenza hemagglutininpolypeptide further comprises a carboxy (C)-terminal truncation startingat an amino acid position from amino acid 532 to amino acid position549, wherein the amino acid positon corresponds to the amino acidposition of SEQ ID NO:1. In certain embodiments, the C-terminaltruncation starts at amino acid position 532, 534, 536, 539, 541, 543,545, 547, or 549, wherein the amino acid position corresponds to theamino acid position of SEQ ID NO:1.

Influenza hemagglutinin (HA) in its native form exists as a trimer onthe cell or virus membrane. In certain embodiments the intracellular andtransmembrane sequence is removed so that a secreted (soluble)polypeptide is produced following expression in cells. Methods toexpress and purify secreted ectodomains of HA have been described (seee.g. Dopheide et al 2009; Ekiert et al 2009, 2011; Stevens et al 2004,2006; Wilson et al 1981). A person skilled in the art will understandthat these methods can also be applied directly to the isolated mutanthemagglutinin polypeptides of the invention in order to achieveexpression of secreted (soluble) polypeptide. Therefore, thesepolypeptides are also encompassed in the invention.

Optionally, a his-tag sequence (HHHHHH (SEQ ID NO: 85) or HHHHHHH (SEQID NO: 86)) may be linked to the (optionally truncated) isolated mutanthemagglutining polypeptide, for purification purposes, optionallyconnected through a linker. Optionally the linker may contain aproteolytic cleavage site to enzymatically remove the his-tag afterpurification.

In certain embodiments, the polypeptides are further stabilized byintroducing a sequence known to form trimeric structures, i.e.GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 87) at the C-terminus ofisolated mutant hemagglutinin polypeptide, optionally connected througha linker. Thus, in certain embodiments, the C-terminal part of theisolated mutant hemagglutinin polypeptide has been replaced by the aminoacid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 87), optionallyconnected through a linker. The linker can contain a cleavage site forprocessing afterwards according to protocols well known to those skilledin the art. To facilitate purification of the soluble form, a tagsequence may be added, e.g. a histidine tag (HHHHHH (SEQ ID NO: 85) orHHHHHHH (SEQ ID NO: 86)) or FLAG tag (DYKDDDDK) (SEQ ID NO: 88) or acombination of these, optionally connected via short linkers. The linkermay optionally contain (part of) a proteolytic cleavage site, e.g., IEGR(SEQ ID NO: 89) (Factor X) or LVPRGS (SEQ ID NO: 90) (thrombin) forprocessing afterwards according to protocols well known to those skilledin the art. The processed proteins are also encompassed in theinvention.

The mutant influenza hemagglutinin polypeptides can be preparedaccording to any technique deemed suitable to one of skill, includingtechniques described below.

Thus, the immunogenic polypeptides of the invention can be synthesizedas DNA sequences by standard methods known in the art and cloned andsubsequently expressed, in vitro or in vivo, using suitable restrictionenzymes and methods known in the art. The present invention thus alsorelates to nucleic acid molecules encoding the above describedpolypeptides. The invention further relates to vectors comprising thenucleic acids encoding the polypeptides of the invention. In certainembodiments, a nucleic acid molecule according to the invention is partof a vector, e.g. a plasmid. Such vectors can easily be manipulated bymethods well known to the person skilled in the art and can, forinstance, be designed for being capable of replication in prokaryoticand/or eukaryotic cells. In addition, many vectors can directly or inthe form of an isolated desired fragment therefrom be used fortransformation of eukaryotic cells and will integrate in whole or inpart into the genome of such cells, resulting in stable host cellscomprising the desired nucleic acid in their genome. The vector used canbe any vector that is suitable for cloning DNA and that can be used fortranscription of a nucleic acid of interest. When host cells are used,it is preferred that the vector is an integrating vector. Alternatively,the vector can be an episomally replicating vector.

The person skilled in the art is capable of choosing suitable expressionvectors and inserting the nucleic acid sequences of the invention in afunctional manner. To obtain expression of nucleic acid sequencesencoding polypeptides, it is well known to those skilled in the art thatsequences capable of driving expression can be functionally linked tothe nucleic acid sequences encoding the polypeptide, resulting inrecombinant nucleic acid molecules encoding a protein or polypeptide inexpressible format. In general, the promoter sequence is placed upstreamof the sequences that should be expressed. Many expression vectors areavailable in the art, e.g. the pcDNA and pEF vector series ofInvitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script fromStratagene, etc, which can be used to obtain suitable promoters and/ortranscription terminator sequences, polyA sequences, and the like. Wherethe sequence encoding the polypeptide of interest is properly insertedwith reference to sequences governing the transcription and translationof the encoded polypeptide, the resulting expression cassette is usefulto produce the polypeptide of interest, referred to as expression.Sequences driving expression can include promoters, enhancers and thelike, and combinations thereof. These should be capable of functioningin the host cell, thereby driving expression of the nucleic acidsequences that are functionally linked to them. The person skilled inthe art is aware that various promoters can be used to obtain expressionof a gene in host cells. Promoters can be constitutive or regulated, andcan be obtained from various sources, including viruses, prokaryotic, oreukaryotic sources, or artificially designed. Expression of nucleicacids of interest can be from the natural promoter or derivative thereofor from an entirely heterologous promoter (Kaufman, 2000). Somewell-known and much used promoters for expression in eukaryotic cellscomprise promoters derived from viruses, such as adenovirus, e.g. theE1A promoter, promoters derived from cytomegalovirus (CMV), such as theCMV immediate early (IE) promoter (referred to herein as the CMVpromoter) (obtainable for instance from pcDNA, Invitrogen), promotersderived from Simian Virus 40 (SV40) (Das et al, 1985), and the like.Suitable promoters can also be derived from eukaryotic cells, such asmethallothionein (MT) promoters, elongation factor 1α (EF-1α) promoter(Gill et al., 2001), ubiquitin C or UB6 promoter (Gill et al., 2001),actin promoter, an immunoglobulin promoter, heat shock promoters, andthe like. Testing for promoter function and strength of a promoter is amatter of routine for a person skilled in the art, and in general canencompass cloning a test gene such as lacZ, luciferase, GFP, etc. behindthe promoter sequence, and test for expression of the test gene. Ofcourse, promoters can be altered by deletion, addition, mutation ofsequences therein, and tested for functionality, to find new,attenuated, or improved promoter sequences. According to the presentinvention, strong promoters that give high transcription levels in theeukaryotic cells of choice are preferred.

The constructs can be transfected into eukaryotic cells (e.g. plant,fungal, yeast or animal cells) or suitable prokaryotic expressionsystems like E. coli using methods that are well known to personsskilled in the art. In some cases a suitable ‘tag’ sequence (such as forexample, but not limited to, a his-, myc-, strep-, or flag-tag) orcomplete protein (such as for example, but not limited to, maltosebinding protein or glutathione S transferase) can be added to thesequences of the invention to allow for purification and/oridentification of the polypeptides from the cells or supernatant.Optionally a sequence containing a specific proteolytic site can beincluded to afterwards remove the tag by proteolytic digestion.

Purified polypeptides can be analyzed by spectroscopic methods known inthe art (e.g. circular dichroism spectroscopy, Fourier TransformInfrared spectroscopy and NMR spectroscopy or X-ray crystallography) toinvestigate the presence of desired structures like helices and betasheets. ELISA, Octet and FACS and the like can be used to investigatebinding of the polypeptides of the invention to the broadly neutralizingantibodies described previously (CR9114, CR8071, CR8033) (Dreyfus etal., Science 337(6100):1343-8 (2012)). Thus, polypeptides according tothe invention having the correct conformation can be selected.

Pharmaceutical/Immunogenic Compositions and Methods of Use

The invention further relates to immunogenic compositions comprising atherapeutically effective amount of at least one of the polypeptidesand/or nucleic acids of the invention. The immunogenic compositionspreferably further comprise a pharmaceutically acceptable carrier. Inthe present context, the term “pharmaceutically acceptable” means thatthe carrier, at the dosages and concentrations employed, will not causeunwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The term “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the composition is administered. Salinesolutions and aqueous dextrose and glycerol solutions can, e.g., beemployed as liquid carriers, particularly for injectable solutions. Theexact formulation should suit the mode of administration. Thepolypeptides and/or nucleic acid molecules preferably are formulated andadministered as a sterile solution. Sterile solutions are prepared bysterile filtration or by other methods known in the art. The solutionscan then be lyophilized or filled into pharmaceutical dosage containers.The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g.pH 5.0 to 7.5.

The invention also relates to influenza mutant hemagglutininpolypeptides, nucleic acid molecules and/or vectors as described abovefor use in inducing an immune response against influenza HA protein. Theinvention also relates to methods for inducing an immune response in asubject, the method comprising administering to a subject, apolypeptide, nucleic acid molecule and/or immunogenic composition asdescribed above. A subject according to the invention preferably is amammal that is capable of being infected with an infectiousdisease-causing agent, in particular an influenza virus, or otherwisecan benefit from the induction of an immune response, such subject forinstance being a rodent, e.g. a mouse, a ferret, or a domestic or farmanimal, or a non-human-primate, or a human. Preferably, the subject is ahuman subject. The invention thus provides methods for inducing animmune response to an influenza virus hemagglutinin (HA) in a subjectutilizing the polypeptides, nucleic acids and/or immunogeniccompositions described herein.

Since it is well known that small proteins and/or nucleic acid moleculesdo not always efficiently induce a potent immune response, it can benecessary to increase the immunogenicity of the polypeptides and/ornucleic acid molecules by adding an adjuvant. In certain embodiments,the immunogenic compositions described herein comprise, or areadministered in combination with, an adjuvant. The adjuvant foradministration in combination with a composition described herein can beadministered before, concomitantly with, or after administration of saidcomposition. Examples of suitable adjuvants include aluminium salts suchas aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, pertussis toxin PT, or tetanus toxoid TT, Matrix M (Isconova).In addition, known immunopotentiating technologies may be used, such asfusing the polypeptides of the invention to proteins known in the art toenhance immune response (e.g. tetanus toxoid, CRM197, rCTB, bacterialflagellins or others) or including the polypeptides in virosomes, orcombinations thereof. Other non-limiting examples that can be used aree.g. disclosed by Coffman et al. (2010).

In an embodiment, the influenza mutant hemagglutinin polypeptides of theinvention are incorporated into viral-like particle (VLP) vectors. VLPsgenerally comprise a viral polypeptide(s) typically derived from astructural protein(s) of a virus. Preferably, the VLPs are not capableof replicating. In certain embodiments, the VLPs can lack the completegenome of a virus or comprise a portion of the genome of a virus. Insome embodiments, the VLPs are not capable of infecting a cell. In someembodiments, the VLPs express on their surface one or more of viral(e.g., virus surface glycoprotein) or non-viral (e.g., antibody orprotein) targeting moieties known to one skilled in the art.

In a specific embodiment, the polypeptides of the invention areincorporated into a virosome. A virosome containing a polypeptideaccording to the invention can be produced using techniques known tothose skilled in the art. For example, a virosome can be produced bydisrupting a purified virus, extracting the genome, and reassemblingparticles with the viral proteins (e.g., the mutant influenzahemagglutinin polypeptides described herein) and lipids to form lipidparticles containing viral proteins.

The invention also relates to the above-described polypeptides, nucleicacids and/or immunogenic compositions for inducing an immune response ina subject against influenza HA, in particular for use as a vaccine. Theinfluenza mutant hemagglutinin polypeptides, nucleic acids encoding suchpolypeptides, or vectors comprising such nucleic acids or polypeptidesdescribed herein thus can be used to elicit protective antibodiesagainst influenza viruses, for example, against the neck or stem domainof the influenza virus hemagglutinin. The invention in particularrelates to polypeptides, nucleic acids, and/or imunogenic compositionsas described above for use as a vaccine in the prevention and/ortreatment of a disease or condition caused by an influenza virus.

The polypeptides of the invention can be used after synthesis in vitroor in a suitable cellular expression system, including bacterial andeukaryotic cells, or alternatively, can be expressed in vivo in asubject in need thereof, by expressing a nucleic acid coding for theimmunogenic polypeptide. Such nucleic acid vaccines may take any form,including naked DNA, plasmids, or viral vectors including adenoviralvectors.

Administration of the polypeptides, nucleic acid molecules, and/orimmunogenic compositions according to the invention can be performedusing standard routes of administration. Non-limiting examples includeparenteral administration, such as intravenous, intradermal,transdermal, intramuscular, subcutaneous, etc, or mucosaladministration, e.g. intranasal, oral, and the like. The skilled personwill be capable to determine the various possibilities to administer thepolypeptides, nucleic acid molecules, and/or immunogenic compositionsaccording to the invention, in order to induce an immune response. Incertain embodiments, the polypeptide, nucleic acid molecule, and/orimmunogenic composition (or vaccine) is administered more than one time,i.e. in a so-called homologous prime-boost regimen. In certainembodiments where the polypeptide, nucleic acid molecule, and/orimmunogenic composition is administered more than once, theadministration of the second dose can be performed after a time intervalof, for example, one week or more after the administration of the firstdose, two weeks or more after the administration of the first dose,three weeks or more after the administration of the first dose, onemonth or more after the administration of the first dose, six weeks ormore after the administration of the first dose, two months or moreafter the administration of the first dose, 3 months or more after theadministration of the first dose, 4 months or more after theadministration of the first dose, etc, up to several years after theadministration of the first dose of the polypeptide, nucleic acidmolecule, and/or immunogenic composition. It is also possible toadminister the vaccine more than twice, e.g. three times, four times,etc, so that the first priming administration is followed by more thanone boosting administration. In other embodiments, the polypeptide,nucleic acid molecule, and/or immunogenic composition according to theinvention is administered only once.

The polypeptides, nucleic acid molecules, and/or immunogeniccompositions can also be administered, either as prime, or as boost, ina heterologous prime-boost regimen.

The invention further provides methods for preventing and/or treating aninfluenza virus disease in a subject utilizing the polypeptides, nucleicacids and/or compositions described herein. In a specific embodiment, amethod for preventing and/or treating an influenza virus disease in asubject comprises administering to a subject in need thereof aneffective amount of a polypeptide, nucleic acid and/or immunogeniccomposition, as described above. A therapeutically effective amountrefers to an amount of the polypeptide, nucleic acid, and/or compositionas defined herein, that is effective for preventing, ameliorating and/ortreating a disease or condition resulting from infection by an influenzavirus. Prevention encompasses inhibiting or reducing the spread ofinfluenza virus or inhibiting or reducing the onset, development orprogression of one or more of the symptoms associated with infection byan influenza virus. Ameloriation as used in herein can refer to thereduction of visible or perceptible disease symptoms, viremia, or anyother measurable manifestation of influenza infection.

Those in need of treatment include those already inflicted with acondition resulting from infection with an influenza virus, as well asthose in which infection with influenza virus is to be prevented. Thepolypeptides, nucleic acids and/or compositions of the invention thuscan be administered to a naive subject, i.e., a subject that does nothave a disease caused by influenza virus infection or has not been andis not currently infected with an influenza virus infection, or tosubjects that already are and/or have been infected with an influenzavirus.

In an embodiment, prevention and/or treatment can be targeted at patientgroups that are susceptible to influenza virus infection. Such patientgroups include, but are not limited to e.g., the elderly (e.g. ≥50 yearsold, ≥60 years old, and preferably ≥65 years old), the young (e.g. ≤5years old, ≤1 year old), hospitalized patients and patients who havebeen treated with an antiviral compound but have shown an inadequateantiviral response.

In another embodiment, the polypeptides, nucleic acids and/orimmunogenic compositions can be administered to a subject in combinationwith one or more other active agents, such as existing, or futureinfluenza vaccines, monoclonal antibodies and/or antiviral agents,and/or antibacterial, and/or immunomodulatory agents. The one or moreother active agents can be beneficial in the treatment and/or preventionof an influenza virus disease or can ameliorate a symptom or conditionassociated with an influenza virus disease. In some embodiments, the oneor more other active agents are pain relievers, anti-fever medications,or therapies that alleviate or assist with breathing.

Dosage regimens of the polypeptides and/or nucleic acid molecules of theinvention can be adjusted to provide the optimum desired response (e.g.,a therapeutic response). A suitable dosage range may for instance be0.1-100 mg/kg body weight, preferably 1-50 mg/kg body weight, preferably0.5-15 mg/kg body weight. The precise dosage of the polypeptides and/ornucleic acid molecules to be employed will e.g. depend on the route ofadministration, and the seriousness of the infection or disease causedby it and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses vary depending on target site, physiological state of the patient(including age, body weight, health), and whether treatment isprophylactic or therapeutic. Usually, the patient is a human, butnon-human mammals, including transgenic mammals can also be treated.Treatment dosages are optimally titrated to optimize safety andefficacy.

The polypeptides of the invention can also be used to verify binding ofmonoclonal antibodies identified as potential therapeutic candidates. Inaddition, the polypeptides of the invention can be used as diagnostictool, for example to test the immune status of an individual byestablishing whether there are antibodies in the serum of suchindividual capable of binding to the polypeptides of the invention. Theinvention thus also relates to an in vitro diagnostic method fordetecting the presence of an influenza infection in a patient saidmethod comprising the steps of a) contacting a biological sampleobtained from said patient with a polypeptide according to theinvention; and b) detecting the presence of antibody-antigen complexes.The polypeptides of the invention can also be used to identify newbinding molecules or improve existing binding molecules, such asmonoclonal antibodies and antiviral agents.

The invention is further illustrated in the following examples andfigures. The examples are not intended to limit the scope of theinvention in any way.

EMBODIMENTS

The invention provides also the following non-limiting embodiments.

Embodiment 1 is an isolated mutant influenza hemagglutinin polypeptidecomprising at least two stabilizing mutations in the polypeptide,wherein the stabilizing mutations comprise substitution mutations at:

-   -   a. amino acid positions 227 and/or 238; and/or    -   b. amino acid positions 384 and/or 476, wherein the amino acid        position corresponds to the amino acid position of SEQ ID NO:1.

Embodiment 2 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 1, wherein

-   -   a. amino acid position 227 is substituted with an amino acid        selected from the group consisting of Q, N, F, I, and Y, and/or        amino acid position 238 is substituted with an amino acid        selected from the group consisting of N, Q, I, and F; and/or    -   b. amino acid position 384 is substituted with an amino acid        selected from the group consisting of W, F, N, Q, and I, and/or        amino acid position 476 is substituted with an amino acid        selected from the group consisting of W, F, Y, I, N, and Q.

Embodiment 3 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 2, wherein

-   -   a. amino acid position 227 is substituted with a Q and amino        acid position 238 is substituted with an I; and/or    -   b. amino acid position 384 is substituted with an I and amino        acid position 476 is substituted with an I.

Embodiment 4 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 1 to 3, further comprising one stabilizingmutation in the polypeptide, wherein the stabilizing mutation is asubstitution at amino acid position 461, wherein the amino acid positioncorresponds to the amino acid position in SEQ ID NO:1.

Embodiment 5 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 4, wherein amino acid position 461 is substituted with anamino acid selected from the group consisting of M, L, W, Y, and R.

Embodiment 6 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 5, wherein amino acid position 461 is substituted with anR.

Embodiment 7 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 3, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:19, SEQ IDNO:35, SEQ ID NO:39 or SEQ ID NO:40.

Embodiment 8 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 6, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence of SEQ ID NO:8.

Embodiment 9 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 1 to 8, further comprising at least oneadditional glycan motif in a head domain of the polypeptide.

Embodiment 10 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 9, wherein the glycan motif comprises a substitution of anamino (N)-linked glycosylation motif in at least one amino acid positionselected from the group consisting of:

-   -   a. 136 or 137,    -   b. 141, and    -   c. 151,        wherein the amino acid position corresponds to the amino acid        position of SEQ ID NO:1.

Embodiment 11 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 10, wherein the glycan motif comprises the substitution ofthe N-linked glycosylation motif at amino acid positions 136 and 141,136 and 151, 137 and 141, 137 and 151, or 141 and 151.

Embodiment 12 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 11, wherein the glycan motif comprises the substitution ofthe N-linked glycosylation motif at amino acid positions 141 and 151.

Embodiment 13 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 10, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, or SEQ ID NO:45.

Embodiment 14 is the isolated mutant influenza hemagglutinin polypeptideof claim any one of embodiments 1 to 13, further comprising a receptorbinding site mutation in the polypeptide.

Embodiment 15 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 14, wherein the receptor binding site mutation comprises asubstitution at an amino acid position selected from the groupconsisting of:

-   -   a. 175,    -   b. 219,    -   c. 257, and    -   d. 258,        wherein the amino acid position corresponds to the amino acid        position of SEQ ID NO:1.

Embodiment 16 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 15, wherein

-   -   a. 175 is substituted with an amino acid selected from the group        consisting of F, W, and Y;    -   b. 219 is substituted with an amino acid selected from the group        consisting of F, W, Y, R, and E;    -   c. 257 is substituted with an amino acid selected from the group        consisting of E, D, V, F; or    -   d. 258 is substituted with an amino acid selected from the group        consisting of E, D, V, and F.

Embodiment 17 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 16, wherein

-   -   a. 175 is substituted with a W,    -   b. 219 is substituted with an E,    -   c. 257 is substituted with an E, or    -   d. 258 is substituted with an E.

Embodiment 18 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 17, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:55, or SEQ ID NO:61.

Embodiment 19 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 14-18, wherein the polypeptide furthercomprises an amino acid substitution at position 136, wherein the aminoacid position corresponds to the amino acid position of SEQ ID NO:1.

Embodiment 20 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 1-19, further comprising a fusion peptideproximal region (FPPR) deletion mutation.

Embodiment 21 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 20, wherein the FPPR deletion mutation comprises adeletion of at least three to seven amino acid residues between aminoacid position 369 and 382, wherein the amino acid position correspondsto the amino acid position of SEQ ID NO:1.

Embodiment 22 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 21, wherein the FPPR deletion mutation comprises adeletion selected from the group consisting of Δ372-376, Δ372-378,Δ373-377, Δ373-376, Δ374-379, Δ374-376, Δ376-380, and Δ377-381.

Embodiment 23 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 22, wherein the FPPR deletion mutation comprises adeletion selected from 4372-376 or 4376-380.

Embodiment 24 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 23, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:62 or SEQ IDNO:68.

Embodiment 25 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 1-24, wherein the mutant hemagglutininpolypeptide comprises an amino acid sequence selected from SEQ ID NO:70,SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84.

Embodiment 26 is an isolated mutant influenza hemagglutinin polypeptidecomprising a fusion peptide proximal region (FPPR) deletion mutation,wherein the FPPR deletion mutation comprises a deletion of at leastthree to seven amino acid residues between amino acid position 369 and382, wherein the amino acid position corresponds to the amino acidposition of SEQ ID NO:1.

Embodiment 27 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 26, wherein the FPPR deletion mutation comprises adeletion selected from the group consisting of Δ372-376, Δ372-378,Δ373-377, Δ373-376, Δ374-379, Δ374-376, Δ376-380, and Δ377-381.

Embodiment 28 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 26 or 27, wherein the FPPR deletion mutation comprises adeletion selected from 4372-376 or 4376-380.

Embodiment 29 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 28, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:62 or SEQ IDNO:68.

Embodiment 30 is an isolated mutant influenza hemagglutinin polypeptidecomprising a receptor binding site mutation in the polypeptide, whereinthe receptor binding site mutation comprises a substitution mutation atan amino acid position selected from the group consisting of:

-   -   a. 175,    -   b. 219,    -   c. 257, and    -   d. 258,        wherein the amino acid position corresponds to the amino acid        position of SEQ ID NO:1.

Embodiment 31 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 30, wherein

-   -   a. 175 is substituted with an amino acid selected from the group        consisting of F, W, and Y;    -   b. 219 is substituted with an amino acid selected from the group        consisting of F, W, Y, R, and E;    -   c. 257 is substituted with an amino acid selected from the group        consisting of E, D, V, F; or    -   d. 258 is substituted with an amino acid selected from the group        consisting of E, D, V, and F.

Embodiment 32 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 31, wherein

-   -   a. 175 is substituted with a W,    -   b. 219 is substituted with an E,    -   c. 257 is substituted with an E, or    -   d. 258 is substituted with an E.

Embodiment 33 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 32, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:55, or SEQ ID NO:61.

Embodiment 34 is the isolated mutant influenza hemagglutinin polypeptideof any of embodiments 30-33, wherein the polypeptide further comprisesan amino acid substitution at amino acid position 136, wherein the aminoacid position corresponds to the amino acid position of SEQ ID NO:1.

Embodiment 35 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 1 to 17, 19 to 23, 26 to 28, 30 to 32, or 34,wherein the mutant influenza hemagglutinin polypeptide comprises aheterologous trimerization domain.

Embodiment 36 is the isolated mutant influenza hemagglutinin polypeptideof any one of claims 1 to 34, wherein the mutant influenza hemagglutininpolypeptide further comprises a carboxy (C)-terminal truncation startingat an amino acid position from amino acid 532 to amino acid position549, wherein the amino acid position corresponds to the amino acidposition of SEQ ID NO:1.

Embodiment 37 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 36, wherein the C-terminal truncation starts at amino acidposition 532, 534, 536, 539, 541, 543, 545, 547, or 549.

Embodiment 38 is the isolated mutant influenza hemagglutinin polypeptideof any one of embodiments 1-37, wherein the mutant influenzahemagglutinin polypeptide further comprises an amino acid substitutionat a cleavage site at amino acid position 362, wherein wherein the aminoacid position corresponds to the amino acid position of SEQ ID NO:1.

Embodiment 39 is the isolated mutant influenza hemagglutinin polypeptideof embodiment 38, wherein amino acid position 362 is substituted with aQ.

Embodiment 40 is an isolated nucleic acid encoding the isolated mutantinfluenza hemagglutinin polypeptide of any one of embodiments 1-39.

Embodiment 41 is a vector comprising the isolated nucleic acid ofembodiment 40.

Embodiment 42 is a host cell comprising the vector of embodiment 41.

Embodiment 43 is a pharmaceutical composition comprising the isolatedmutant influenza hemagglutinin polypeptide of any one of embodiments1-39 and a pharmaceutically acceptable carrier.

Embodiment 44 is a pharmaceutical composition comprising the isolatednucleic acid of embodiment 40.

Embodiment 45 is a pharmaceutical composition comprising the vector ofembodiment 41.

Embodiment 46 is a method of inducing an immune response against aninfluenza virus in a subject in need thereof, the method comprisingadministering to the subject in need thereof the pharmaceuticalcomposition of any one of embodiments 43 to 45.

Embodiment 47 is a method of producing an isolated mutant influenzahemagglutinin polypeptide, the method comprising culturing the host cellof embodiment 42 under conditions capable of producing the mutantinfluenza hemagglutinin polypeptide and recovering the mutant influenzahemagglutinin polypeptide from the cell or culture.

Embodiment 48 is a method of producing the pharmaceutical composition ofembodiment 43, the method comprising combining the isolated mutantinfluenza polypeptide with a pharmaceutically acceptable carrier.

EXAMPLES

TABLE 1 Standard amino acids, abbreviations and properties Side chainSide chain charge Amino Acid 3-Letter 1-Letter polarity (pH 7.4) alanineAla A nonpolar Neutral arginine Arg R polar Positive asparagine Asn Npolar Neutral aspartic acid Asp D polar Negative cysteine Cys C nonpolarNeutral glutamic acid Glu E polar Negative glutamine Gln Q polar Neutralglycine Gly G nonpolar Neutral histidine His H polar Positive (10%)/Neutral (90%) isoleucine Ile I nonpolar Neutral leucine Leu L nonpolarNeutral lysine Lys K polar Positive methionine Met M nonpolar Neutralphenylalanine Phe F nonpolar Neutral proline Pro P nonpolar Neutralserine Ser S polar Neutral threonine Thr T polar Neutral tryptophan TrpW nonpolar Neutral tyrosine Tyr Y polar Neutral valine Val V nonpolarNeutral

Example 1: Stem Based Polypeptides—Structure and Design Elements

The structure and location of alterations in the sequence of thepolypeptides representing the ectodomain of influenza virushaemagglutinin (HA₀) are shown in FIG. 1A. When expressed as a solubleectodomain, the polypeptides were carboxy (C)-terminally truncated; e.g.at position 536 of SEQ ID NO:1, as it is noted that for UFV180846, SEQID NO:2, the polypeptide is only 535 amino acids) omitting the nativeC-terminal transmembrane and cytosolic domain (amino acids 550-585). Itis noted that for the numbering of the amino acid positions, the WildType HA B/Brisbane/60/08 (SEQ ID NO:1) numbering was used and includedthe signal peptide (residues 1-15).

To stabilize HA, increase the expression, and ensure correct folding andtrimerization similar to the parental wild-type full-length HA,substitutions were introduced in the polypeptides at positions 227, 238,384, 461, and 476 (FIGS. 1A-1B).

To improve the HA stem epitope accessibility for broadly bindingantibodies, e.g. mAb CR9114 (as described in WO2013/007770), the lengthof the flexible loop comprising the fusion proximal region (FPPR, aminoacids 362-382) was reduced by a about 5 amino acids in certainpolypeptides.

To obstruct receptor or antibody binding to the solvent exposed surfacesof the HA head domain in certain polypeptides, the conserved receptorbinding site (RBS) was substituted (Q257E) and/or one or more aminoacids residues were substituted to introduce the amino (N)-linkedglycosylation motif (NxS/T, whereas x is not a P, i.e. at positions 136,141 and 151) or alternatively substituted to a charged residue (i.e. atpositions 136 and 257).

The polypeptides can be resistant to trypsin like protease cleavage bysubstituting the natural monobasic cleavage site amino acid arginine (R)at position 362 (FIG. 1B) into, e.g. glutamine (Q). In contrast tonative pre-fusion HA, polypeptides of the invention including the R329Qsubstitution are trypsin like protease resistant and cannot be cleavedanymore. Without cleavage into HA₁ and HA₂ the influenza virushemagglutinin protein is unable to undergo conformational changes to thepost-fusion state and can subsequently not mediate viral fusion.

Example 2: Characterization of Stabilizing Mutations Designs

The soluble polypeptides represented the influenza virus hemagglutinin(HA) of influenza Type B. Multiple residue substitutions with the aim tostabilize and improve the folding of the polypeptides were tested atposition 461 (FIG. 2B), at positions 227 and 238 (FIG. 2C), and atpositions 384 and 476 (FIG. 2D). Expression and folding of thepolypeptides were assessed in Expi293F cell culture supernatant.

Protein Expression in Mammalian Cells

DNA fragments encoding the polypeptides were synthesized (Genscript;Piscataway, NJ) and cloned in the pcDNA2004 expression vector (modifiedpcDNA3 plasmid with an enhanced CMV promotor).

The polypeptides contained a carboxy (C)-terminal foldon trimerizationdomain (except for UFV171348, SEQ ID NO:39 and UFV171387, SEQ ID NO:40)and a FLAG-Linker-His tag for screening purposes and purification. Theywere produced in the eukaryotic suspension cell line Expi293F at microscale (200 μL). In short, cells were transiently transfected withindustrial grade DNA in 96-halfdeepwell plates (System Duetz) at a celldensity of 2.5×10E+06 vc/mL using the ExpiFectamine 293 transfection kit(Gibco, ThermoFisher Scientific; Waltham, MA) and incubated in Expi293Expression Medium (Gibco, ThermoFisher Scientific) at 37° C., 250 rpm,8% CO₂ and 75% humidity. Cell culture supernatants containing secretedpolypeptides were harvested at day 3 and clarified by centrifugation (10minutes at 400×g) followed by filtration (96-well Filter plates, 0.22 μmPVDF membrane, Corning; Corning, NY).

Culture Supernatant Analysis

Expression and folding of the polypeptides were assessed by amplifiedluminescent proximity homogeneous assay (AlphaLISA, FIGS. 2B-2D)according to the manufacturer's instructions (PerkinElmer; Waltham, MA).This in-solution and in-binding-equilibrium assay is based on successfulbinding of both a donor and acceptor bead to the polypeptides viaspecific antibodies. When in close proximity, laser irradiation of thedonor bead at 680 nm generated a flow of singlet oxygen, triggeringchemical events in a nearby acceptor bead, resulting in chemiluminescentemission at 615 nm. Expression levels were measured via theExpression-AlphaLISA setup by simultaneous addition of Nickel donorbeads (that binds/complexes with the His tag) and beads coupled to anantibody directed against the FLAG tag to the cell culture supernatant.This Expression-AlphaLISA setup recognized the C-terminalFLAG-Linker-His tag irrespective of the folding of the polypeptides. Thecorrect folding of the polypeptides was assessed in a Binding-AlphaLISAby simultaneous addition of Nickel donor beads, human IgG antibodyCR9114 (as described in WO2013/007770) at a concentration of 2 nM, andanti-human IgG acceptor beads to the cell culture supernatant. A signalwas only obtained if the polypeptide correctly folded and permitted thebinding of the influenza virus HA specific IgGs.

For all AlphaLISA setups, the detector beads were added at aconcentration of 10 μg/mL. The culture supernatants were tested atdifferent dilutions to avoid the hook-effect according to themanufacturer's instructions. Readout was performed 2 hours afterincubation at room temperature in the dark using the EnSight™ multimodeplate reader (PerkinElmer). Data were normalized to reference constructUFV170090 (SEQ ID NO:3), wild type HA B/Brisbane/60/08 (SEQ ED NO:1)including a foldon trimerization domain and a FLAG-Linker-His tag, thatwas set to 100%.

The thermo-stability of the polypeptides was determined by DifferentialScanning Fluorimetry (DSF) by monitoring the fluorescent emission ofadded Sypro Orange Dye (ThermoFisher Scientific) to the culturesupernatant. Upon gradual increase of the temperature, from 25° C. to95° C. (60° C. per hour), the polypeptides unfolded, and the fluorescentdye bound to the exposed hydrophobic residues leading to acharacteristic change in emission. The melting curves were measuredusing a ViiA7 real time PCR machine (Applied Biosystems; Foster City,CA), and the Tm₅₀ values were calculated by the Spotfire suite (TibcoSoftware Inc.; Palo Alto, CA). The Tm₅₀ values represent the temperatureat which 50% of the protein is unfolded and thus are a measure for thetemperature stability of the polypeptides.

The content of the expressed polypeptides in the Expi293F cell cultureharvests was assessed by analytical Size Exclusion Chromatography (SEC)in an Ultra High-Performance Liquid Chromatography (UHPLC) using aVanquish system (ThermoFisher Scientific) with a BEH 200A column(Waters, injection volume 40 μL, flow 0.35 mL/min.). The elution wasmonitored by a Helios light scattering detector (Wyatt Technology;Goleta, CA). The SEC profiles were analyzed by the Astra 6 softwarepackage (Wyatt Technology).

Results and Conclusion

Most of the alternative amino acids at position 461 were well tolerated,except for the Tryptophan (UFV171702, SEQ ID NO:6) that resulted in adecrease of −40% mAb CR9114 binding of (FIG. 2B). Polypeptide UFV171741(SEQ ID NO:8) that included an arginine residue at position 461displayed −2.8-fold increase in mAb CR9114 binding. Substitution of theresidue at position 227 resulted in an −1.5-fold increase of CR9114binding for the amino acids tested, whereas substitutions at position238 did not affect antibody binding (FIG. 2C). A combination of aglutamine and isoleucine at these positions, respectively, resulted in asignificant increase of CR9114 binding (˜2.5-fold). Introducingsubstitutions to position 384 resulted in a 3 to 4-fold decrease inCR9114 binding, whereas substitutions at position 476 were welltolerated or resulted in a modest increase in binding levels (UFV170550(SEQ ID NO:29) and UFV170551 (SEQ ID NO:30)). Polypeptides with bothresidues substituted displayed a significant decrease in CR9114 binding,except when both residues were substituted to an isoleucine. Thiscombination (UFV170556 (SEQ ID NO:35)) displayed a −2.3-fold increase inCR9114 binding (FIG. 2C). Temperature stability of the polypeptide, asdetermined by DSF, indicated a 3.9° C. increase in Tm₅₀ uponintroduction of the Q227 and 1238 substitutions (UFV170525 (SEQ IDNO:19) vs UFV170090 (SEQ ID NO:3), whereas the expression level was notsignificantly affected (FIG. 2D).

No noteworthy effect of substitutions I384 and I476 on the temperaturestability was observed (0.5° C. decrease), however, the polypeptideincluding these mutations (UFV170556 (SEQ ID NO:35)) displayed anincreased expression level (˜1.4-fold) and increased binding of CR9114(˜2-fold). Removal of the foldon trimerization domain also resulted inan increase in expression level (UFV171348 (SEQ ID NO:39)) versusUFV170556 (SEQ ID NO:35)), however, a decrease in CR9114 binding wasobserved. The combination of all four (4) favorable substitutions atpositions 227, 238, 384, and 476 resulted in a polypeptide thatexpressed well (2-fold increase to reference) and bound CR9114 well(˜1.7-fold increase to reference). Strikingly, the polypeptide was verystable (Tm₅₀ of 64.7° C., 5.2° C. higher than parent construct) andexpressed as a soluble trimeric polypeptide in the absence of a foldontrimerization domain (FIG. 2F).

Taken together the results showed that by substituting four (4) residuesin the core of the HA, a polypeptide was generated that formed solubletrimers in the absence of heterologous trimerization domains, whichrepresent a correctly folded and stable pre-fusion conformation of wildtype influenza B HA.

Example 3: Characterization of Added N-Linked Glycosylation Motifs tothe Head Domain Designs

At various positions N-linked glycosylation motifs NxT/S (wherein x isnot a P) were introduced to the head domain of the polypeptides. Forpositions 136, 137, and 151 an N and T were introduced, whereas forposition 141 an asparagine was present in the wild type sequence and themotif was completed by introducing a threonine at position 143 (FIG.3A).

Culture Supernatant Analysis

DNA fragments encoding the polypeptides of the invention weresynthesized as described in Example 2. The polypeptides including aFLAG-Linker-His tag for screening purposes and purification wereproduced in the eukaryotic suspension cell line Expi293F at micro scale(200 μL). UFV171472 (SEQ ID NO:43) was expressed with a Foldontrimerization domain, the other polypeptides were expressed without aFoldon trimerization domain.

Expression and folding of the polypeptides of the invention wereassessed by AlphaLISA as described in Example 2. Binding of CR8071(Dreyfus et al., Science 337(6100):1343-8 (2012)) and SD84 (Laursen etal., Science 362(6414):598-602 (2018)) was performed at a concentrationof 1.5 nM and 2 nM respectively. The trimer-AlphaLISA setup was used todetermine the content of trimeric polypeptides present in the culturesupernatant. The trimer-AlphaLISA assay relied on human IgGs such as46B8C (WO2015/148806A1), which specifically bound to monomeric HA. If a1:1 mix of differently labeled 46B8C (biotin or DIG labeled) was addedto HA, an AlphaLISA signal was only be detected if a multimer,permitting binding of at least two antibodies, was present. It was shownpreviously that this trimer-AlphaLISA setup preferably detected trimersand was insensitive for dimers, multimers or monomers. Trimer-AlphaLISAwas performed by simultaneous addition of Streptavidin donor beads andanti-DIG IgG acceptor beads to the culture supernatant in the presenceof biotinylated- and DIG-labelled 46B8C IgGs (each at 1 nM). Data forpolypeptides UFV171991 (SEQ ID NO:45), UFV171992 (SEQ ID NO:44), andUFV171993 (SEQ ID NO:42) were normalized to reference constructUFV171990 (SEQ ID NO:41), representing a stabilized B/Brisbane/60/08 HA.For polypeptide UFV171472 (SEQ ID NO:43), data was normalized toreference construct UFV170090 (SEQ ID NO:3), wild type HAB/Brisbane/60/08 including a Foldon trimerization domain. Referenceconstructs were set to 100%.

Results and Conclusion

The introduction of additional N-linked glycosylation motifs to the headdomain of the polypeptides of the invention at positions 136, 137, 141,or 151 was possible (FIG. 3B) and only a minimal decrease in expressionlevels were observed (up to ˜30% for UFV171472 (SEQ ID NO:43)). Bindingof stem (CR9114) and neck (CR8071) specific antibodies was maintained,whereas an expected decrease in head domain specific binder SD84 wasobserved. The highest reduction in SD84 binding was observed forpolypeptides with a N-linked glycan introduction at position 137(UFV171472, SEQ ID NO:43) or at position 151 (UFV171991, SEQ ID NO:45).The head-binding of SD84 was reduced by 40% and 52% respectivelyrelative to the polypeptide without additional N-linked glycosylationsites.

Example 4: Characterization of Receptor Binding Site ModificationsDesigns

To reduce the affinity of the conserved receptor binding to its naturalligand sialic acid and to alter the conserved epitope for head-bindingantibodies in and around the receptor binding site, point substitutionswere introduced to the polypeptides disclosed herein. At position 175,alternative hydrophobic residues were introduced, whereas bothhydrophobic and charged residues were evaluated for positions 219, 257,and 258 (FIG. 4A).

Culture Supernatant Analysis

DNA fragments encoding the polypeptides were synthesized as described inExample 2. The polypeptides including a FLAG-Linker-His tag forscreening purposes were produced in the eukaryotic suspension cell lineExpi293F at micro scale (200 μL). Expression and folding of thepolypeptides were assessed by AlphaLISA as described in Examples 2 and3. Data was normalized to reference construct UFV171990 (SEQ ID NO:41),representing a stabilized B/Brisbane/60/08 HA including FLAG-Linker-Histag, that was set to 100%.

Results and Conclusion

Compared to the reference, all polypeptides with altered residues nearor in the receptor binding site displayed reduced expression levels(FIG. 4B). Substitutions at positions 219 and 258 are tolerated least;UFV172072 (SEQ ID NO:54) and UFV172064 (SEQ ID NO:46) were the lowest(11%) and highest (56%) expressed polypeptides. Substitutions atpositions 175 and 257 were accepted better with regard to proteinexpression. Polypeptides UFV172073 (SEQ ID NO:55), UFV172075 (SEQ IDNO:57), and UFV172078 (SEQ ID NO:60) were minimally affected and reach alevel of 75%, 70%, and 74% relative to the reference. Similarly, thesethree polypeptides displayed the highest trimer content; 90%, 89%, and78% respectively. Furthermore, binding of stem binding monoclonalantibody CR9114 was preserved (71-94%), while binding of head domainspecific SD84 was considerably altered; UFV172073 (SEQ ID NO:55) hardlybound (17%), while UFV172075 (SEQ ID NO:57) displayed a drastic increasein binding (579%). For UFV172078, with a substitution at position 175(SEQ ID NO:60), binding of SD84 was only minimally affected (74%).

Overall, substitutions in and around the receptor binding site were notwell tolerated. Polypeptides including the 257E, 257V, or 175Wsubstitution displayed a small but acceptable decrease in expressionlevel, trimer content, and binding of mAb CR9114. The only observeddifference with these polypeptides was with binding of SD84.

Example 5: Characterization of Fusion Peptide Proximal Region (FPPR)Deletions in the Polypeptides of the Invention Designs

The residues of the structurally undefined loop following the HA₀cleavage site at position 362 was referred to as the Fusion PeptideProximal Region (FPPR, residues 369-383, FIG. 1A and FIG. 5A).Polypeptides comprising an FPPR deletion of varying length, from 3 to 7residues, and position were evaluated with the aim to increase the HAstability and accessibility of conserved stem epitopes.

Culture Supernatant Analysis

DNA fragments encoding the polypeptides were synthesized as described inExample 2. The polypeptides including a FLAG-Linker-His tag forscreening purposes were produced in the eukaryotic suspension cell lineExpi293F at micro scale (200 μL). Expression and folding of thepolypeptides were assessed by AlphaLISA as described in Examples 2 and3. Data was normalized to reference construct UFV171990 (SEQ ID NO:41),representing a stabilized B/Brisbane/60/08 HA including FLAG-Linker-Histag, that was set to 100%.

Results and Conclusion

Partial deletions of the FPPR did not alter the protein expressionlevels notably (83-110%, FIG. 5B). For two polypeptides, UFV172680 (SEQID NO:63) and UFV172683 (SEQ ID NO:65), a −2-fold decrease in trimerformation was observed, whereas all other polypeptides showed similartrimer content compared to the reference. Larger differences wereobserved for the binding of stem specific mAb CR9114. With 24% CR9114binding compared to the reference, UFV172690 (SEQ ID NO:69) showed thelowest binding, which suggested that deletions beyond position 380 werenot well tolerated. UFV172680 (SEQ ID NO:63), UFV172683 (SEQ ID NO:65),and UFV172691 (SEQ ID NO:68) also displayed reduced CR9114 binding with55%, 66%, and 74%, respectively, compared to reference. Binding of neckspecific mAb CR8071 was only minimally affected, and the relativebinding was within the range of 67% to 104% compared to reference.Binding of head domain specific SD84 displayed a larger spread inbinding; UFV172683 (SEQ ID NO:65) showed with a 7 amino acid deletion,the lowest binding (43%), and UFV172686 (SEQ ID NO:66) displayed thehighest binding (167%).

Overall, partial deletions of the FPPR were well tolerated; expressionlevel, trimer formation and correct folding were maintained or showed aminimal decrease, even if up to 7 amino acids of this highly conservedloop were removed. Folding of CR9114 was clearly impaired when deletionsreach position 381, which was presumably too close to the conserved HAstem epitopes. Polypeptides UFV172680 (SEQ ID NO:63) and UFV172683 (SEQID NO:65) showed decreased binding for all assessed antibodies.

Example 6: Alternative Truncations at the C-Terminus Designs

Hemagglutinin is a membrane protein that is located at the surface ofthe viral particles and infected cells with the C-terminal part of theprotein embedded in the viral membrane. For the soluble versions of thepolypeptides, the transmembrane domain was deleted by a truncation atthe start of the transmembrane domain (TM). Additionally, alternativetruncation positions were evaluated in the stabilized HAB/Brisbane/60/08 reference polypeptide UFV180284 (SEQ ID NO:70). Whereasthe reference polypeptide was expressed without a Foldon trimerizationdomain and including a C-tag, the variants with alternative C-terminaltruncations were expressed as tag-free soluble trimeric polypeptides(Table 2).

TABLE 2 Alternative C-terminal truncations of the polypeptides derivedfrom the ectodomain of HA from B/Brisbane/60/08. C-terminus of HAectodomain 531 532 533 534 535 536 537 538 539 540 541 B/Brisbane/ S L NI T A A S L N D 60/08 UFV180454 S L N I T A A S L N D UFV180455 S L N IT A A S L N D UFV180456 S L N I T A A S L N D UFV180457 S L N I T A A SL N D UFV180458 S L N I T A A S L N D UFV180459 S L N I T A A S L — —UFV180460 S L N I T A — — — — — UFV180461 S L N I — — — — — — —UFV180462 S L — — — — — — — — — C-terminus of HA ectodomain TM 542 543544 545 546 547 548 549 550 551 552 B/Brisbane/ D G L D N H T I L L Y60/08 UFV180454 D G L D N H T I — — — UFV180455 D G L D N H — — — — —UFV180456 D G L D — — — — — — — UFV180457 D G — — — — — — — — —UFV180458 — — — — — — — — — — — UFV180459 — — — — — — — — — — —UFV180460 — — — — — — — — — — — UFV180461 — — — — — — — — — — —UFV180462 — — — — — — — — — — — “—” indicates the truncated residuesbetween positions 533 and 552. “TM” stands for trans-membrane domain.Putative N-glycan sites are highlighted at amino acid position 533 and546.

Culture Supernatant Analysis

DNA fragments encoding the polypeptides listed in Table 2 weresynthesized and expressed in EXPI-293 cell cultures as described inExample 2 and 4. The harvested culture supernatants were analyzed forthe presence of expressed trimeric polypeptide by analytical SEC usingHPLC as described in Example 2.

Results and Conclusion

Analysis of the culture supernatants by SEC indicated one major peak(˜6.5-minute retention time) for all tested constructs that correspondedto the trimeric form of the polypeptide (FIG. 6 ). Minimal effect of thealternative C-terminal truncations on the expression level of thetrimeric polypeptides was observed; only a minor decrease in trimer peakheight was observed for UFV180461 (SEQ ID NO:78) and UFV180462 (SEQ IDNO:79), −20% in peak height compared to reference UFV180284 (SEQ IDNO:70). Furthermore, a gradual increase in retention time in the sizeexclusion column was observed, which correlated with the decrease inpolypeptide trimer size upon the stepwise truncation of the C-terminus.Likely the change in retention time was enhanced by the removal of twoputatively N-linked glycosylated asparagine's at positions 533 and/or546.

In summary, C-terminal truncations between residue 533 and 549 of thepolypeptides of the invention were well tolerated and only minor effectson expression levels of the trimeric influenza B HAs were observed.

Example 7: Expression, Purification and In Vitro Characterization ofTrimeric Polypeptides of the Invention Designs

To characterize the combination of additional N-linked glycosylationmotifs, receptor binding site (RBS) substitutions, and deletions in thefusion peptide proximal region, they were introduced in stabilized HAB/Brisbane/60/08 in the absence of a Foldon trimerization domain. Inpolypeptides without the introduced N-linked glycosylation motif atposition 136 a glutamate (E) was introduced; UFV180137 (SEQ ID NO:82),UFV180251 (SEQ ID NO:83), and UFV180284 (SEQ ID NO:84). The RBSsubstitution (257E) was included in all polypeptides. For comparisonpurpose, WT HA B/Brisbane/60/08 including C-terminal Foldontrimerization domain (UFV170088: SEQ ID NO:80) was used. Allpolypeptides were produced in ExpiCHO cells including a C-tag, afour-residue acid peptide (E-P-E-A) fused to the C-terminus of thepolypeptides

Protein Expression and Purification

DNA fragments encoding the polypeptides were synthesized (Genscript) andcloned in the pcDNA2004 expression vector (modified pcDNA3 plasmid withan enhanced CMV promotor). The polypeptides were produced in ExpiCHOsuspension cells cultured in ExpiCHO™ expression medium by transienttransfection of respective industrial grade DNA (≤0.01 EU/μg endotoxinlevel and ≥90% supercoil content) using ExpiFectamine™ transfectionreagent (Gibco, ThermoFisher Scientific) according to the manufacturer'sprotocol. ExpiFectamine CHO Enhancer and ExpiCHO Feed (Gibco,ThermoFisher Scientific) were added to the cell cultures 1-day posttransfection according to the manufacturer's protocol. Culturesupernatants containing the secreted polypeptides were harvested at day10 and clarified by centrifugation, followed by filtration over a 0.2 μmbottle top filter (Corning). The polypeptides were expressed at mediumscale (˜70 mL) and larger scale (˜350 mL).

The polypeptides were purified by means of a two-step protocol. First,the harvested and clarified culture supernatant (large scaletransfection) was loaded on a HiScale 16/20 column (GE Healthcare;Chicago, IL) packed with an affinity resin

(Capture Select; ThermoFisher Scientific) that consisted of a C-tagspecific single domain antibody, immobilized on Agarose based bead(ThermoFisher Scientific). This resin was highly specific for bindingproteins with the C-tag. The amount of applied polypeptide in theharvested culture supernatant was determined by OCTET (anti C-tag) priorto purification. Elution of the C-tagged proteins was performed using aTRIS buffer containing 2M MgCl₂. Based on the UV signal (A280) theeluted fractions were pooled and filtered through a Millex-GV 0.22 μmfilter membrane (MilliporeSigma; Burlington, MA). Subsequently, thecollected elution peak was applied to a Superdex 200 pg 26/60 column (GEHealthcare) equilibrated in running buffer (20 mM Tris, 150 mM NaCl,pH7.8) for polishing purpose, i.e., to remove the minimal amount ofmultimeric and monomeric protein. Based on the UV signal (A280) thetrimer fractions were pooled.

Culture Supernatant and Purified Protein Analysis

The level of expressed polypeptide in the cell culture supernatant wasassessed by Bio-Layer Interferometry using the OCTET platform accordingto the manufacturer's instructions (FortéBio; Fremont, CA). First astandard curve was established, using Streptavidin biosensors(FortéBio), loaded with CaptureSelect™ Biotin anti C-tag conjugate(ThermoFisher Scientific), by assessing the binding shift of a dilutionseries of a well-defined reference batch of purified homologouspolypeptide (stabilized HA B/Brisbane/60/08 including C-terminal C-tag;UFV172551 SEQ ID NO:96). Subsequently, the binding shift of pre-diluted(in kinetics buffer, FortéBio) cell culture supernatants containing thepolypeptides was measured and the concentration of the polypeptides wascalculated using the established standard curve

The trimer content of the polypeptides in the culture supernatant and ofpurified polypeptide was assessed by Size Exclusion Chromatography MultiAngle Light Scattering (SEC-MALS) analysis using a High PerformanceLiquid Chromatography (HPLC) Infinity 1260 series setup (Agilent; SantaClara, CA). Of each purified polypeptide 40 μg was run (1 mL/min.) overa TSK gel G3000SWxl column (Sigma-Aldrich; St. Louis, MO) and the molarmass of the eluted material was measured by a miniDAWN Treos Multi AngleLight Scattering detector and Optilab T-rEx differential refractometer(Wyatt Technology). The data were analyzed by the Astra 6 softwarepackage (Wyatt Technology) and molecular weight calculations werederived from the refractive index signal.

The antigenicity of purified polypeptides was assessed by ELISA (EC₅₀values of the antibody binding). To this end, polypeptides were coatedat a concentration of nM and incubated with a dilution series ofmonoclonal antibody (mAb) CR9114 (as described in WO2013/007770), CR8071(as described in Dreyfus et al., 337(6100):1343-8 (2012)), SD84 (asdescribed in (Laursen et al., Science 362(6414):598-602 (2018)), and34B5 (as described in WO2015/148806). A starting concentration of 70 nMwas applied for CR9114, CR8071, and 34B5, whereas a startingconcentration of 100 nM was used for SD84. Antibody binding wasdetermined by incubation with a secondary antibody, anti-human Fc HRP(Mouse anti-Human IgG, Jackson ImmunoResearch; West Grove, PA) andvisualized by addition of POD substrate. Read out was performed usingthe EnSight™ multimode plate reader (PerkinElmer; Waltham, MA). The EC₅₀values were calculated using the Spotfire suite (Tibco Software Inc.;Palo Alto, CA).

The thermo-stability of the polypeptides was determined in the culturesupernatant by Differential Scanning Fluorimetry (DSF) as described inExample 2 by monitoring the fluorescent emission of Sypro Orange Dye(ThermoFisher Scientific) added to a 6 μg polypeptide solution.

Results and Conclusion

The analysis of crude cell culture supernatant by SEC-MALS (FIG. 7A,left panel) indicated the presence of predominantly soluble trimericpolypeptides (˜7 minutes retention time). Similar analysis alsoindicated that the two-step purification protocol yielded pure trimericpolypeptide (FIG. 7A, right panel). Furthermore, the trimericpolypeptides expressed at a high level as determined by OCTET; up to a2-fold increase was observed compared to the reference (FIG. 7B). Thepurified polypeptides were correctly folded as evident by ELISA analysisshowing strong CR9114 binding to the stem of the polypeptide (with EC₅₀values in the lower nanomolar range (<2.6 nM)). Similarly, EC₅₀ valueswere observed for binding of neck specific mAb CR8071. In contrast, nobinding was observed for the head-domain specific binder SD84. Likelythe introduced N-linked glycosylation motifs were glycosylated andprevented SD84 from binding due to steric hindrance by the glycanmoieties. The temperature at which 50% of the polypeptide unfolds wasdetermined by DSF. All polypeptides were temperature stable anddisplayed Tm₅₀ values of 68.3° C., 69.2° C., 69.4° C., and 69.3° C. for,respectively, UFV180131 (SEQ ID NO:81), UFV180137 (SEQ ID NO:82),UFV180251 (SEQ ID NO:83), and UFV180284 (SEQ ID NO:84). The Tm₅₀ valuefor the reference, wild type HA including a Foldon trimerization domain,was ˜8.6° C. lower, which indicated the combination of substitutions anddeletions had a significant effect on the temperature stability of thepolypeptide. Using an alternative C-terminal truncation position andknocking out the HA₀ cleavage site resulted in a decrease in proteinexpression level; however, each polypeptide expressed well at a highlevel that is comparable to the reference (FIG. 7C). Polypeptide foldingwas not affected and EC₅₀ values in the lower nanomolar range wereobserved (≤4.6 nM) for stem specific mAb CR9114 and neck specific mAbCR8071 binding. Similar to what was observed for SD84, no binding ofhead-domain specific mAb172498 (WO2015/148806) was observed. Likely, theintroduced N-linked glycosylation motifs were glycosylated and preventedbinding of mAb172498 due to steric hindrance by the glycan moieties. Allpolypeptides displayed similar Tm₅₀ values compared to the polypeptideswith a non-mutated cleavage site and other truncation positions.

In summary, the combination of stabilizing substitutions and fusionpeptide proximal region deletion was beneficial and allowed the additionof non-native head domain glycans and receptor binding sitesubstitutions. The polypeptides expressed well, were purified from thecell culture supernatant as properly folded trimeric polypeptides, weretemperature stable, and maintained the proper HA folding and trimericpre-fusion conformation in solution.

Example 8: Expression of Soluble Stabilized Influenza B HA Compared toWild Type Influenza B HA in Various Subtypes Designs

In addition to the HA polypeptides described in Example 2-7, furtherstabilized Influenza B HAs were expressed and compared to theirrespective wild type soluble HA ectodomains. Thus, a glutamine (Q) atposition 227, isoleucine (I) at position 238, isoleucine (I) at position384, arginine (R) at position 461, and an isoleucine (I) at position 476were introduced in the HA amino acid sequences of four additionalInfluenza B strains: B/Lee/1940, B/Yamagata/16/1988 (Yamagata lineage),B/Florida/04/2006 (Yamagata lineage), and B/Iowa/06/2017 (Victorialineage). All polypeptide included the fusion peptide proximal region(FPPR) deletion mutation 372-376 except for the B/Iowa/06/2017 derivedpolypeptide. Expression levels of the polypeptides in Expi293F cellculture supernatant, three days after transfection, were compared to therespective WT polypeptides without the mutations.

Culture Supernatant Analysis

DNA fragments encoding the polypeptides of the invention weresynthesized as described in Example 2. The wild type polypeptidesincluding a His-tag and the stabilized polypeptides including alinker-sortase recognition sequence-His tag for screening purposes andpurification, were produced in the eukaryotic suspension cell lineExpi293F at micro scale (200 μL).

The level of expressed polypeptide in the cell culture supernatant wasassessed by Bio-Layer Interferometry using the OCTET platform(FortéBio). In short, a standard curve was established using anti-HIS(HIS2) Biosensors (FortéBio) by measuring the binding shift of adilution series of a well-defined reference batch of a purifiedcomparable polypeptide. Subsequently, the binding shifts of pre-diluted(in kinetics buffer, FortéBio) cell culture supernatants containing thepolypeptides of the invention were measured and the concentration of thepolypeptides was calculated using the established standard curve.

The presence of the expressed polypeptides and its quaternary structure(which indicates whether the polypeptide is a monomer, trimer ormultimer) in the culture supernatant was assessed by Size ExclusionChromatography Multi Angle Light Scattering (MALS) in an UltraHigh-Performance Liquid Chromatography (UHPLC) setup using a Vanquishsystem (ThermoFisher Scientific). For the B/Lee/40, B/Yamagata/16/1988,and B/Florida/04/2006 derived polypeptides a BEH 200A column (Water,injection volume 40 μL, flow 0.35 mL/min) was used, for theB/Iowa/06/2017 derived polypeptide a Unix-C 300A column (SepaxTechnologies, injection volume 15 μL, flow 0.1 mL/min) was used. Theelution was monitored by a Helios light scattering detector (WyattTechnologies). The SEC profiles were analyzed by the Astra 6 softwarepackage (Wyatt Technology).

Result and Conclusion

Like observed in Example 2, substitution to glutamine at position 227,and isoleucine's at positions 238, 384 and 476, an arginine at position461 with or without the deletion of the FPPR (residues 372-376) in thewild type HA of different strains resulted in an increase in expressionas determined by Bio-Layer Interferometry (FIG. 8A). The analysis of thecrude cell culture supernatant by SEC-MALS (FIG. 8B) showed that uponintroduction of the stabilizing mutations, for all soluble stabilizedHAs a distinct trimer (T) peak appears at a retention time −6.5 minuteswhich is higher than the trimer peaks observed for the respective wildtype HA ectodomains (FIG. 8 .B). Furthermore, none of the stabilized HAdisplayed a monomer (M) peak as appears at a retention time between 6.5and 7 minutes for the wild type B/Iowa/06/2017 HA.

In summary, the data confirm that introduction of mutations 227Q, 238I,384I, 461R, and 476I result in an increased expression and formation ofstable soluble trimeric HA polypeptides.

REFERENCES

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SEQUENCES SEQ ID NO 1: Full length B/Brisbane/60/08MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL SEQ ID NO 2: UFV180846MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSNHTVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAEPEASEQ ID NO 3: UFV170090MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 4: UFV171700MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELMVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 5: UFV171701MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELLVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 6: UFV171702MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELWVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 7: UFV171703MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELYVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 8: UFV171741MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 9: UFV170519MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 10: UFV170520MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQNFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 11: UFV170521MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQFFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 12: UFV170522MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQIFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 13: UFV170523MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQYFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 14: UFV170515MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTNYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 15: UFV170516MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTQYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 16: UFV170517MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 17: UFV170518MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTFYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 18: UFV170524MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTNYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 19: UFV170525MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 20: UFV170541MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWWGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 21: UFV170542MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWFGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 22: UFV170543MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWNGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 23: UFV170544MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWQGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 24: UFV170545MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 25: UFV170546MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEWLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 26: UFV170547MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEFLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 27: UFV170548MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEYLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 28: UFV170549MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 29: UFV170550MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDENLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 30: UFV170551MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEQLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 31: UFV170552MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWWGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEWLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 32: UFV170553MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWWGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEFLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 33: UFV170554MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWFGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEWLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 34: UFV170555MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWWGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEYLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 35: UFV170556MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 36: UFV170557MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWNGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDENLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 37: UFV170558MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWQGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEQLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 38: UFV170559MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWNGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEQLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 39: UFV171348MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 40: UFV171387MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 41: UFV171990MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 42: UFV171993MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSNHTVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 43: UFV171472MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTNNTINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSHHHHHHSEQ ID NO 44: UFV171992MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINATNAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 45: UFV171991MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 46: UFV172064MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKFYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 47: UFV172065MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKWYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 48: UFV172066MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKYYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 49: UFV172067MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKRYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 50: UFV172068MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKEYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 51: UFV172069MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQEGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 52: UFV172070MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQDGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 53: UFV172071MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQVGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 54: UFV172072MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQFGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 55: UFV172073MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 56: UFV172074MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPDSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 57: UFV172075MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPVSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 58: UFV172076MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPFSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 59: UFV172077MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAFPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 60: UFV172078MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAWPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 61: UFV172079MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAYPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 62: UFV172678MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 63: UFV172680MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 64: UFV172681MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 65: UFV172683MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 66: UFV172686MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 67: UFV172687MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 68: UFV172691MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 69: UFV172690MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIAAADYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSHHHHHH SEQ ID NO 70: UFV180284MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIEPE ASEQ ID NO 71: UFV180454MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTISEQ ID NO 72: UFV180455MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHSEQ ID NO 73: UFV180456MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDSEQ ID NO 74: UFV180457MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGSEQ ID NO 75: UFV180458MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDSEQ ID NO 76: UFV180459MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLSEQ ID NO 77: UFV180460MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASEQ ID NO 78: UFV180461MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNISEQ ID NO 79: UFV180462MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLSEQ ID NO 80: UFV170088MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITASGSLVPRGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA SEQ ID NO 81: UFV180131MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSNHTVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIEPEASEQ ID NO 82: UFV180137MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIEPEASEQ ID NO 83: UFV180251MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIEPEASEQ ID NO 84: UFV180846MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSNHTVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAEPEASEQ ID NO 85: HIS-Tag HHHHHH SEQ ID NO 86: HIS-Tag HHHHHHHSEQ ID NO 87: Trimerization Domain GYIPEAPRDGQAYVRKDGEWVLLSTFLSEQ ID NO 88: FLAG Tag DYKDDDDKSEQ ID NO 89: Factor X Proteolytic Cleavage Site IEGRSEQ ID NO 90: Thrombin Proteolytic Cleavage Site LVPRGSSEQ ID NO 91: UFV180847MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAEPEASEQ ID NO 92: UFV180848MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAEPEASEQ ID NO: 93 UFV180849MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAEPEASEQ ID NO 94: Yamagata lineage (B/Florida/04/06)MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGSFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMLAIFIVYMVSRDNVSCSICL SEQ ID NO 95: ConsensusMKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTETRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKNGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKKLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTISEQ ID NO 96: UFV172551MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTIGGSEPEA SEQ ID NO 97: UFV180846-secretedDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSNHTVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITA SEQ ID NO 98: UFV180847-secretedDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITA SEQ ID NO 99: UFV180848-secretedDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITA SEQ ID NO 100: UFV180949-secretedDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSEHNVINATNAPGGPYNITTSGSCPNITNGNGFFATMAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPESGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEQGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITA SEQ ID NO 101: UFV180567MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTRSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNIPSAKVSILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINAETAPGGPYKVGTSGSCPNVANRNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMNGLHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAHHHHHHSEQ ID NO 102: UFV180566MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTRSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNIPSAKVSILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINAETAPGGPYKVGTSGSCPNVANRNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQQFTSSANGVTTIYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMNGLHDEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITASGSLVPSGSLPETGGGSHHHHH HSEQ ID NO 103: UFV180565MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPNCLNCTDLDVALGRPMCMGTIPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTHNVINAERAPGGPYRLGTSGSCPNVTSRNGFFATMAWAVPRDNKTATNPLTVEVPYICTKGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGDFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAHHHHHHSEQ ID NO 104: UFV180400MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPNCLNCTDLDVALGRPMCMGTIPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTHNVINAERAPGGPYRLGTSGSCPNVTSRNGFFATMAWAVPRDNKTATNPLTVEVPYICTKGEDQITVWGFHSDDKTQMKNLYGDSNPQQFTSSANGVTTIYVSQIGDFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITASGSLVPSGSLPETGGGSHHHHHHSEQ ID NO 105: UFV180571MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGSFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAHHHHHHSEQ ID NO 106: UFV180570MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQQFTSSANGVTTIYVSQIGSFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITASGSLVPSGSLPETGGGSHHHHH HSEQ ID NO 107: UFV190909MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINAEGAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPDKNKTATNPLTIEVPYVCTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDKIAAGTFDAGEFSLPTFDSLNITAHHHHHHSEQ ID NO 108: UFV190521MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINAEGAPGGPYKIGTSGSCPNITNGNGFFATMAWAVPDKNKTATNPLTIEVPYVCTEGEDQITVWGFHSDNETQMAKLYGDSKPQQFTSSANGVTTIYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWIGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELRVLLSNEGIINSEDEILLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDKIAAGTFDAGEFSLPTFDSLNITASGSLPETGGGSHHHHHH

1. An isolated mutant influenza hemagglutinin polypeptide comprising atleast two stabilizing mutations in the polypeptide, wherein thestabilizing mutations comprise substitution mutations at: a. amino acidpositions 227 and/or 238; and/or b. amino acid positions 384 and/or 476,wherein the amino acid position corresponds to the amino acid positionof SEQ ID NO:1.
 2. The isolated mutant influenza hemagglutininpolypeptide of claim 1, wherein a. amino acid position 227 issubstituted with an amino acid selected from the group consisting of Q,N, F, I, and Y, and/or amino acid position 238 is substituted with anamino acid selected from the group consisting of N, Q, I, and F; and/orb. amino acid position 384 is substituted with an amino acid selectedfrom the group consisting of W, F, N, Q, and I, and/or amino acidposition 476 is substituted with an amino acid selected from the groupconsisting of W, F, Y, I, N, and Q.
 3. The isolated mutant influenzahemagglutinin polypeptide of claim 2, wherein a. amino acid position 227is substituted with a Q and amino acid position 238 is substituted withan I; and/or b. amino acid position 384 is substituted with an I andamino acid position 476 is substituted with an I.
 4. The isolated mutantinfluenza hemagglutinin polypeptide of any one of claims 1 to 3, furthercomprising one stabilizing mutation in the polypeptide, wherein thestabilizing mutation is a substitution at amino acid position 461,wherein the amino acid position corresponds to the amino acid positionin SEQ ID NO:1.
 5. The isolated mutant influenza hemagglutininpolypeptide of claim 4, wherein amino acid position 461 is substitutedwith an amino acid selected from the group consisting of M, L, W, Y, andR.
 6. The isolated mutant influenza hemagglutinin polypeptide of claim5, wherein amino acid position 461 is substituted with an R.
 7. Theisolated mutant influenza hemagglutinin polypeptide of claim 3, whereinthe mutant influenza hemagglutinin polypeptide comprises an amino acidsequence selected from SEQ ID NO:19, SEQ ID NO:35, SEQ ID NO:39, or SEQID NO:40.
 8. The isolated mutant influenza hemagglutinin polypeptide ofclaim 6, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:
 108. 9.The isolated mutant influenza hemagglutinin polypeptide of any one ofclaims 1 to 8, further comprising at least one additional glycan motifin a head domain of the polypeptide.
 10. The isolated mutant influenzahemagglutinin polypeptide of claim 9, wherein the glycan motif comprisesa substitution of an amino (N)-linked glycosylation motif in at leastone amino acid position selected from the group consisting of: a. 136 or137, b. 141, and c. 151, wherein the amino acid position corresponds tothe amino acid position of SEQ ID NO:1.
 11. The isolated mutantinfluenza hemagglutinin polypeptide of claim 10, wherein the glycanmotif comprises the substitution of the N-linked glycosylation motif atamino acid positions 136 and 141, 136 and 151, 137 and 141, 137 and 151,or 141 and
 151. 12. The isolated mutant influenza hemagglutininpolypeptide of claim 11, wherein the glycan motif comprises thesubstitution of the N-linked glycosylation motif at amino acid positions141 and
 151. 13. The isolated mutant influenza hemagglutinin polypeptideof claim 10, wherein the mutant influenza hemagglutinin polypeptidecomprises an amino acid sequence selected from SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, or SEQ ID NO:45.
 14. The isolated mutant influenzahemagglutinin polypeptide of claim any one of claims 1 to 13, furthercomprising a receptor binding site mutation in the polypeptide.
 15. Theisolated mutant influenza hemagglutinin polypeptide of claim 14, whereinthe receptor binding site mutation comprises a substitution at an aminoacid position selected from the group consisting of: a. 175, b. 219, c.257, and d. 258, wherein the amino acid position corresponds to theamino acid position of SEQ ID NO:1.
 16. The isolated mutant influenzahemagglutinin polypeptide of claim 15, wherein a. 175 is substitutedwith an amino acid selected from the group consisting of F, W, and Y; b.219 is substituted with an amino acid selected from the group consistingof F, W, Y, R, and E; c. 257 is substituted with an amino acid selectedfrom the group consisting of E, D, V, F; or d. 258 is substituted withan amino acid selected from the group consisting of E, D, V, and F. 17.The isolated mutant influenza hemagglutinin polypeptide of claim 16,wherein a. 175 is substituted with a W, b. 219 is substituted with an E,c. 257 is substituted with an E, or d. 258 is substituted with an E. 18.The isolated mutant influenza hemagglutinin polypeptide of claim 17,wherein the mutant influenza hemagglutinin polypeptide comprises anamino acid sequence selected from SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:55, or SEQ ID NO:61.
 19. The isolated mutant influenza hemagglutininpolypeptide of any one of claims 14-18, wherein the polypeptide furthercomprises an amino acid substitution at position 136, wherein the aminoacid position corresponds to the amino acid position of SEQ ID NO:1. 20.The isolated mutant influenza hemagglutinin polypeptide of any one ofclaims 1-19, further comprising a fusion peptide proximal region (FPPR)deletion mutation.
 21. The isolated mutant influenza hemagglutininpolypeptide of claim 20, wherein the FPPR deletion mutation comprises adeletion of at least three to seven amino acid residues between aminoacid position 369 and 382, wherein the amino acid position correspondsto the amino acid position of SEQ ID NO:1.
 22. The isolated mutantinfluenza hemagglutinin polypeptide of claim 21, wherein the FPPRdeletion mutation comprises a deletion selected from the groupconsisting of Δ372-376, Δ372-378, Δ373-377, Δ373-376, Δ374-379,Δ374-376, Δ376-380, and Δ377-381.
 23. The isolated mutant influenzahemagglutinin polypeptide of claim 22, wherein the FPPR deletionmutation comprises a deletion selected from 4372-376 or 4376-380. 24.The isolated mutant influenza hemagglutinin polypeptide of claim 23,wherein the mutant influenza hemagglutinin polypeptide comprises anamino acid sequence selected from SEQ ID NO:62 or SEQ ID NO:68.
 25. Theisolated mutant influenza hemagglutinin polypeptide of any one of claims1-24, wherein the mutant hemagglutinin polypeptide comprises an aminoacid sequence selected from SEQ ID NO:70, SEQ ID NO:81, SEQ ID NO:82,SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,SEQ ID NO: 102, SEQ ID NO: 104, or SEQ ID NO:
 106. 26. An isolatedmutant influenza hemagglutinin polypeptide comprising a fusion peptideproximal region (FPPR) deletion mutation, wherein the FPPR deletionmutation comprises a deletion of at least three to seven amino acidresidues between amino acid position 369 and 382, wherein the amino acidposition corresponds to the amino acid position of SEQ ID NO:1.
 27. Theisolated mutant influenza hemagglutinin polypeptide of claim 26, whereinthe FPPR deletion mutation comprises a deletion selected from the groupconsisting of Δ372-376, Δ372-378, Δ373-377, Δ373-376, Δ374-379,Δ374-376, Δ376-380, and Δ377-381.
 28. The isolated mutant influenzahemagglutinin polypeptide of claim 26 or 27, wherein the FPPR deletionmutation comprises a deletion selected from Δ372-376 or Δ376-380. 29.The isolated mutant influenza hemagglutinin polypeptide of claim 28,wherein the mutant influenza hemagglutinin polypeptide comprises anamino acid sequence selected from SEQ ID NO:62 or SEQ ID NO:68.
 30. Anisolated mutant influenza hemagglutinin polypeptide comprising areceptor binding site mutation in the polypeptide, wherein the receptorbinding site mutation comprises a substitution mutation at an amino acidposition selected from the group consisting of: a. 175, b. 219, c. 257,and d. 258, wherein the amino acid position corresponds to the aminoacid position of SEQ ID NO:1.
 31. The isolated mutant influenzahemagglutinin polypeptide of claim 30, wherein a. 175 is substitutedwith an amino acid selected from the group consisting of F, W, and Y; b.219 is substituted with an amino acid selected from the group consistingof F, W, Y, R, and E; c. 257 is substituted with an amino acid selectedfrom the group consisting of E, D, V, F; or d. 258 is substituted withan amino acid selected from the group consisting of E, D, V, and F. 32.The isolated mutant influenza hemagglutinin polypeptide of claim 31,wherein a. 175 is substituted with a W, b. 219 is substituted with an E,c. 257 is substituted with an E, or d. 258 is substituted with an E. 33.The isolated mutant influenza hemagglutinin polypeptide of claim 32,wherein the mutant influenza hemagglutinin polypeptide comprises anamino acid sequence selected from SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:55, or SEQ ID NO:61.
 34. The isolated mutant influenza hemagglutininpolypeptide of any of claims 30-33, wherein the polypeptide furthercomprises an amino acid substitution at amino acid position 136, whereinthe amino acid position corresponds to the amino acid position of SEQ IDNO:1.
 35. The isolated mutant influenza hemagglutinin polypeptide of anyone of claim 1 to 17, 19 to 23, 26 to 28, 30 to 32, or 34, wherein themutant influenza hemagglutinin polypeptide comprises a heterologoustrimerization domain.
 36. The isolated mutant influenza hemagglutininpolypeptide of any one of claims 1 to 34, wherein the mutant influenzahemagglutinin polypeptide further comprises a carboxy (C)-terminaltruncation starting at an amino acid position from amino acid position532 to amino acid position 549, wherein the amino acid positioncorresponds to the amino acid position of SEQ ID NO:1.
 37. The isolatedmutant influenza hemagglutinin polypeptide of claim 36, wherein theC-terminal truncation starts at amino acid position 532, 534, 536, 539,541, 543, 545, 547, or
 549. 38. The isolated mutant influenzahemagglutinin polypeptide of any one of claims 1-37, wherein the mutantinfluenza hemagglutinin polypeptide further comprises an amino acidsubstitution at a cleavage site at amino acid position 362, whereinwherein the amino acid position corresponds to the amino acid positionof SEQ ID NO:1.
 39. The isolated mutant influenza hemagglutininpolypeptide of claim 38, wherein amino acid position 362 is substitutedwith a Q.
 40. An isolated nucleic acid encoding the isolated mutantinfluenza hemagglutinin polypeptide of any one of claims 1-39.
 41. Avector comprising the isolated nucleic acid of claim
 40. 42. A host cellcomprising the vector of claim
 41. 43. A pharmaceutical compositioncomprising the isolated mutant influenza hemagglutinin polypeptide ofany one of claims 1-39 and a pharmaceutically acceptable carrier.
 44. Apharmaceutical composition comprising the isolated nucleic acid of claim40.
 45. A pharmaceutical composition comprising the vector of claim 41.46. A method of inducing an immune response against an influenza virusin a subject in need thereof, the method comprising administering to thesubject in need thereof the pharmaceutical composition of any one ofclaims 43 to
 45. 47. A method of producing an isolated mutant influenzahemagglutinin polypeptide, the method comprising culturing the host cellof claim 42 under conditions capable of producing the mutant influenzahemagglutinin polypeptide and recovering the mutant influenzahemagglutinin polypeptide from the cell or culture.
 48. A method ofproducing the pharmaceutical composition of claim 43, the methodcomprising combining the isolated mutant influenza polypeptide with apharmaceutically acceptable carrier.